2008 Nsf Career_proposal Only

Project Summary The long-term mission of our laboratory is to enable and make important discoveries in molecular cell biology by innovating new biophysical methods and culturing interdisciplinary research and education partnerships. The specific research goals of this 5-year proposal are to develop new methods for single-molecule analysis of DNA and chromatin extracted from living yeast cells, and thus open a new research area that combines the powers of singlemolecule and genetic approaches. Our long-term mission is also supported by our specific educational and broader impacts goals in this proposal, which will multiply the impact of our research goals. These include participation in Open Science, integration of research and university education, and community and local school outreach. Together, these research and education goals in this proposal will establish a successful career path for this PI as leader of an exciting biophysics research laboratory collaborating with leading chromatin and transcription biologists, successful university educator, and recruiter of underrepresented minorities to research careers. Intellectual Merit The unifying themes of our research plan are single-molecule analysis and chromatin biology. One of the main goals we are pursuing is to develop a method based on DNA unzipping with optical tweezers that will map proteins on native chromatin with much higher precision and sensitivity than the currently most powerful technique—chromatin Immunoprecipitation (ChIP). We are proposing three separate but synergistic research goals: 1. Site-specific single-molecule analysis of yeast chromatin remodeling during RNA Polymerase II elongation. Mapping with high resolution along a gene the positions of histones and polymerases on individual chromatin fibers. 2. Single-molecule analysis of DNA resection and chromatin remodeling during DNA double-strand break repair in yeast. 3. Stochastic simulation of polymerase and other enzyme kinetics, in silico DNA unzipping simulation, and development of new single-molecule analysis tools Broader Impacts 1. Open Science—Sharing of research data, research protocols and teaching material on OpenWetWare. Recruitment and training of new open scientists. 2. Integration of research with undergraduate and graduate education. Incorporation of exciting biophysics results into Physics 102 “Physics for non-scientists” course. Mentoring of graduate students in Physics 102 lecturing. Transforming “Junior Lab” course into an open science course and developing new biophysics experimental modules. 3. Community and school outreach in Albuquerque. PI participation in outreach and development of new partnerships with middle school teachers. PI encouragement of strong participation by graduate and undergraduate researchers in lab.

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Project Description Career Mission The long-term mission of our laboratory is to enable important discoveries in molecular cell biology by innovating new biophysical methods and culturing interdisciplinary research and education partnerships. The specific research goals of this 5-year proposal are to develop new methods for single-molecule analysis of DNA and chromatin extracted from living yeast cells, and thus open a new research area that combines the powers of single-molecule and genetic approaches. Our long-term mission is also supported by our specific educational and broader impacts goals in this proposal, which will multiply the impact of our research goals. These include participation in Open Science, integration of research and university education, and community and local school outreach. Together, these research and education goals in this proposal will establish a successful career path for this PI as leader of an exciting biophysics research laboratory collaborating with leading chromatin and transcription biologists, successful university educator, and recruiter of underrepresented minorities to research careers. Research Goals 1. Site-specific single-molecule analysis of yeast chromatin during RNA Polymerase II elongation 2. Single-molecule analysis of DNA and chromatin structure in yeast during doublestrand break repair 3. Stochastic simulation of polymerase kinetics, in silico unzipping, and development of new single-molecule analysis tools

Education & Broader Impacts Goals 1. Open Science—Sharing of research data, research protocols, teaching material, and recruitment of new open scientists via OpenWetWare 2. Integration of research with undergraduate and graduate education 3. Community and school outreach in Albuquerque, NM

Optical Research Overview nucleosome Trap DNA in eukaryotic cells exists as chromatin, which is repeating units of DNA wrapped around histone proteins. These DNARNA Pol II histone units are called nucleosomes, and play a fundamental ssDNA role in both positive and negative regulation of proteins that require access to the DNA code. Cells have a variety of enzymes Coverglass that can modify the structure of the chromatin by moving, Figure 1 Proposed unzipping of single removing, or adding histones, or by modifying specific amino acid chromatin fibers with optical tweezers. residues on the histones. This chromatin remodeling affects the Monitoring the length of ssDNA and the ability of other proteins to access the DNA and has a profound unzipping forces will reveal the position of nucleosomes and polymerases with impact on critical processes such as DNA repair and gene close to base pair resolution. transcription by RNA polymerase. Understanding of these dynamic processes is currently hampered by the inability to characterize with high spatial and temporal resolution the changes to chromatin inside living cells. Therefore, we are developing biophysical tools with single-molecule sensitivity to address this need. One of the main goals we are pursuing is to develop a single-molecule method (see Fig. 1) for mapping proteins on chromatin that will far surpass the capabilities of the currently most powerful technique—chromatin Immunoprecipitation (ChIP). We are also pursuing other goals for analysis of genomic DNA and chromatin with optical and magnetic tweezers and nanostructured devices. The unifying themes of our research goals are

single-molecule analysis and chromatin biology, and we are seeking to build a career foundation in a new arena of single-molecule biophysics applied to in vivo systems. The field of single-molecule (SM) biophysics has already made a profound impact on many areas of molecular cell biology, in two general areas, single-molecule detection (SMD)(1-14) and single-molecule manipulation (SMM) (15-59)(60-63). The majority of high impact achievements in both SMD and SMM have been studies of in vitro systems developed from purified components, e.g. single-molecule in vitro biochemistry. Recently, achievements have been made in SMD inside living cells(10, 64) and the need is clearly seen for the ability to detect and track single-molecules in real-time in living cells. We view a parallel set of crucial goals as the application of SMM for analysis of material inside living cells or extracted from the living environment. Many of the existing examples of SM analysis of molecules extracted from living cells have involved chromatin(34, 65-68). The studies have demonstrated the value of this line of experiments, but as of yet, no methods have been developed for site-specific SM chromatin analysis. The wealth of potential discoveries in this area remains for the most part untapped, and is the main focus of our CAREER proposal. Project 1: Site-specific single-molecule analysis of yeast chromatin during RNA Polymerase II elongation 1.1 Background and open questions During elongation by Pol II, histones are transiently evicted to allow passage of the polymerase and reassembled in the wake of the polymerase to prevent transcription from cryptic initiation sites. Concomitant with these changes in nucleosome structure, various lysine residues of the histone tails are modified via acetylation, methylation, and ubiquitylation, and the functions of these modifications are not yet well understood(69). One modification is the monoubiquitylation of histone H2B, which has been associated with active transcription in both yeast and humans(70, 71). In addition to histone marks, the nucleosome dynamics during transcription are regulated by various histone chaperones which facilitate proper assembly of nucleosomes. One of these chaperones is Spt16, a subunit of the FACT complex, which is known to stimulate Pol II transcription(72-75). Recent data have shown that Spt16 and H2B ubiquitylation interact to facilitate proper Pol II transcription, though many questions remain as to the nature of this interaction(76-78). Single-molecule unzipping (SMU) will provide information unattainable with current methods As shown in Table 1.1, we are proposing to use single-molecule unzipping techniques(54-56, 79, 80) to answer current questions about the interplay of H2B ubiquitylation with the FACT chaperone and how they together affect transcription elongation by Pol II(76, 78). In contrast to ChIP methods, which provide an ensemble average and cannot provide detailed information about a single molecule, our methods will allow mapping of Pol II and nucleosome locations with much higher resolution (3 base pairs versus 100’s base pairs) than permitted by ChIP, and detection of multiple Pol II and nucleosome complexes on the same single DNA molecule. This may reveal direct correlations between molecules that are not decipherable with ChIP measurements—for example, in the K123R mutant compared with WT, do the Pol II molecules appear to be stalled directly upstream of nucleosomes? Additionally, the unzipping force pattern for Pol II may be distinctly different depending on the transcriptional orientation of the complex— thus providing a mechanism for detecting Pol II complexes undergoing antisense transcription. Proper chromatin structure is essential for suppressing cryptic initiation(69, 81), and in an spt16 mutant that displays significant levels of cryptic initiation(77), a Pol II molecule that is loaded in the antisense orientation is bound to collide with an upstream Pol II engaged in proper gene transcription. A head-on collision would likely serve as an obstacle to productive transcription, and may explain depleted downstream Pol II occupancy.

Table 1.1 Summary single-molecule investigation of chromatin remodeling and Pol II transcription in yeast Cell type

Selected features seen with ensemble assays

New information possibly obtained via singlemolecule unzipping experiments

Wild Type Yeast

Anti-correlation of histone and Pol II occupancy

Higher resolution information than available with ChIP regarding nucleosome and Pol II positions. Correlations between nucleosomes and Pol II on single chromatin molecules.

Information about Pol II elongation rate and processivity from ChIP H2B K123R Yeast (No H2B ubiqtuitin)

Slightly higher H3 at GAL1 ORF; histone reassembly delay

Possible change in correlation of Pol II and nucleosome positions, if there is an increased nucleosomal barrier to Pol II.

Pol II levels reduced by 50% at GAL1 spt16ts Yeast 37C FACT defect; Reassembly defect

Lower levels of H2B ubiquitylation Pol II levels reduced by 50% at GAL1, but no increase in histone occupancy Significant cryptic initiation at STE11, FLO8, and other genes

A difference in nucleosome unzipping signal upon switch from 30 to 37C, reflecting structural difference in nucleosomes. Identify cryptically initiated Pol II with antisense orientation, impeding sense-oriented Pol II.

spt16/K123R Double mutant of above

Compared with WT, less histone and Pol II occupancy on GAL1 ORF Increased cryptic initiation relative to spt16 alone Rapid loss of Pol II on GAL1 ORF after galactose to glucose switch MNase shows disordered chromatin

Confirm stretches of naked DNA implied by low histone and Pol II occupancy; identify improperly assembled nucleosomes. Identify antisense-oriented Pol II as above with spt16 mutant. High resolution Pol II mapping to confirm rapid elongation or high termination rate.

1.2 Single-molecule unzipping method and evidence for future success We are pursuing the development of innovative single-molecule methods for analyzing site-specific native chromatin isolated from living cells. The key innovation required is to be able to create singlemolecule tethers from chromatin isolated from living cells. With this innovation, we will have the ability to apply sophisticated single-molecule analysis to chromatin extracted from living cells at a specific time (relative to a cellular event, such as a DSB) and a specific location in the genome (see Table 1.2). Table 1.2 New capabilities enabled by innovative single-molecule method for analysis of chromatin from living cells Expected Capabilities

Explanation

Mapping of Pol II and nucleosome binding with close to base pair resolution. Absolute determination of occupancy (no need to normalize signal, as is necessary with ChIP)

Published data showed ability to locate a mononucleosome to within 3 base pairs. Can count all proteins on a single molecule and thus directly measure occupancy. In ChIP, signal must be relative—e.g. H3 occupancy in mutant relative to wild type.

Determination of the location of Pol II and nucleosomes on a singlemolecule with high relative position

Published data show very distinct signal for mononucleosome unzipping. Expect distinct signal for Pol II. Will calibrate with stalled in vitro transcription complexes from Adelman lab (NIH NIEHS).

Able to study chromatin from exact location in genome

Specificity by purification and ligation of known sticky end from HO, IPpoI, or I-SceI endonuclease.

Genome-wide shotgun mapping of chromatin

Naked DNA unzipping force pattern is easily predicted from known sequence. Exact genomic location of a fragment determined by cross-correlating unzipping force pattern with library of simulated unzipping force patterns for all possible endonuclease sites.

Chromatin dynamics at specific sites in the genome are routinely studied with ChIP and other methods, which accounts for much of the current knowledge of Pol II elongation and interaction with FACT and H2Bub. Important work has also been done with single-molecule analysis of reconstituted chromatin(2, 33, 37, 38, 52, 63, 82) and chromatin extracted from living cells(34, 65-68), but to date, single-molecule has not been combined with site-specific native chromatin as we are proposing. Our idea, as shown in Figure 1 (above), is to map with high resolution the positions of histones and polymerases on single chromatin fibers, by unzipping the fibers with optical tweezers. We are uniquely suited to pursue this project, because in A graduate school (in M. D. Wang lab at Cornell Physics) the GAL1 UAS GAL-YLR454w PI was the innovator of the method for mapping proteinI-SceI I-SceI ~10 kb DNA interactions by unzipping single DNA molecules with B optical tweezers(54, 55). The PI designed a versatile Dig Nick Biotin I-SceI 5’ 3’ “unzipping construct” (Fig. 1.1 B) which provided the complementary 5’ necessary basis for unzipping tethers (a dig label, a single Figure 1.1 (A) GAL1-inducible YLR454w gene will nick in the DNA, and a biotin label) and a method for be engineered with two flanking I-SceI sites, or alternatively an I-SceI and a Not I site, to allow for adapting the sticky end for ligation to a variety of releasing ~10 kb soluble fragments (B) The versatile (54, 55, 60, 62, 83) downstream unzipping segments . We are unzipping construct from Koch et al. 2002 will be used, the only change being inclusion of the I-SceI leveraging this versatility in this project, where now the complementarity. downstream unzipping segments will be native chromatin fragments isolated from living yeast cells. Essentially, the ligation step that links the in vitro unzipping construct to the specifically digested chromatin is the point where traditional single-molecule experiments will meet traditional yeast genetics. Once we have achieved this key step, the unzipping experiments will proceed essentially the same as previously with wholly in vitro systems(60). We additionally will have opened the door for a variety of other single-molecule analysis capabilities for native chromatin and DNA (see research section 2 and 3).

Strong evidence exists for ability to map nucleosomes by unzipping

Force (pN)

We have several reasons to be A B Data from Shundrovsky, Koch, et al., Biophys J 83, p. 1098 40 et al., Nature Structural confident that if we can create single& Molecular Biology 13, p. 549 (Not Koch Data) molecule tethers from genomic EcoRI binding chromatin, then we will be able to 30 produce and interpret valuable data regarding the position of nucleosomes 20 on the chromatin. This confidence is best illustrated in Fig. 1.2, where the ©2006 Nature Publishing Group 10 unzipping signal is shown for a variety 500 700 900 1100 1300 Number of base pairs unzipped of different protein-DNA complexes. In Figure 1.2 (A) Koch data from Koch et al. 2002, demonstrating proof -ofFig. 1.2 A, the purple trace shows the principle single-molecule protein-DNA mapping by unzipping. Seven total EcoRI-DNA interactions detected at the expect locations (bars under x-axis). unzipping signal for EcoRI binding to The binding couplets separated by 11 basepairs easily distinguished. (B) Not DNA (in the absence of Mg++). These Koch Data: from Shundrovsky et al. 2006, demonstrated repeatable unzipping data were taken with the optical signal from octamer easily distinguished from H3 -H4 tetramer. Mapped mononucleosome position with about 3 bp accuracy. tweezers that Koch constructed in the Wang lab at Cornell and using the DNA molecule that Koch constructed and that contained repeats of EcoRI binding couplets (marked by solid vertical lines under the x-axis)(55). Binding signals are easily seen at the expect EcoRI binding sites. In Fig. 1.2 B, reproduced from(60), three separate unzipping traces are shown—naked DNA, a mononucleosome (octamer), and monotetrasome. These data were taken with the same optical tweezers as in A, modifying the sticky end of the unzipping construct in Fig.

1.1 B to ligate an assembled nucleosome unzipping segment. As with the EcoRI model system, vertical increases in force are seen when protein-DNA contacts impede DNA unzipping.

We believe we will be able to map polymerase positions by unzipping Unlike with nucleosomes, there is not yet any published data illustrating the forces encountered when unzipping through a stable Pol II complex. Therefore, we do not yet know what Pol II signatures to look for when unzipping native chromatin, and we do not even know whether repeatable and sensible signatures will exist. Given the beautiful force signatures that in vitro nucleosomes showed(60), the fact that Pol II elongating complexes are highly stable, and that crystal structures can be obtained for elongating Pol II complexes(84, 85) we anticipate that Pol II will show identifiable unzipping force signatures. In order to lay the groundwork, we will first unzip Pol II elongation complexes that have been prepared in vitro by the Adelman lab (NIH NIEHS) via nucleotide starvation after initiation. Dr. Adelman’s group will provide us with stalled complexes and defined 5’ overhangs either upstream or downstream from the elongation complex. We will ligate these complexes onto our versatile unzipping construct (Fig. 1.1) and seek to identify repeatable patterns in the unzipping forces. Due to the asymmetric nature of the elongation complex (compared with the symmetric nucleosome complex), we anticipate a difference between unzipping from the downstream versus upstream direction: and thus we may be able to distinguish sense from anti-sense oriented Pol II complexes in vivo. In addition to providing valuable reference data for our analysis of native chromatin, this project will also lay the foundation for investigation of Pol II in vitro transcription via purified components that we will pursue with the Adelman lab and their collaborators via funding in addition to the CAREER award. 1.3 Isolation of native chromatin and preparation for unzipping Intact chromatin fragments are routinely isolated from living yeast cells in ChIP assays. However, these methods have the potentially undesirable step of fixing the cells with formaldehyde, which forms proteinprotein and protein-DNA crosslinks. (It is possible that the crosslinking will not inhibit our unzipping and may even help, but we want to avoid this complication initially.) In ChIP assays, these crosslinks are reversed via incubation at 65C, but this step also destroys the chromatin structure. There are methods for Native ChIP (NChIP)(86), but these methods too are not desirable as they require non-specific nuclease digestion of the chromatin to mono or di-nucleosomes (thus obscuring correlations between nucleosomes on single fibers). The two most promising protocols for isolation of specific regions of native chromatin suitable for single-molecule unzipping are the recombinase circles method(87, 88) and the plasmid minichromosome method(89). The Griesenbeck et al. recombinase method is appealing, but due to the relative ease of transforming yeast with plasmids and existing connections with Dave Clark (NIH), we are currently working to adapt this method. Our goal is to investigate the role of FACT and H2Bub during elongation by Pol II, and the YLR454 gene is an attractive system for doing so(77, 78, 90) because it is a long gene (~8 kb) and we can look at potentially several polymerases and nucleosomes on a given individual isolated chromatin fiber. However, the length is not ideal for the plasmid system, and so we are initially working with a plamsid containing the PHO5 promoter. This system has well characterized nucleosomes and nucleasehypersensitive sites and the nucleosomes are reliably evicted upon induction of PHO5(91, 92). To date, we have obtained this plasmid from the authors, engineered an I-SceI site in hypersensitive site upstream of PHO5, and transformed the plasmid into yeast. We are currently verifying via Southern blot that we can digest chromatin in isolated nuclei with I-SceI and other enzymes.(91, 92) Next, we will isolate chromatin similar to the minichromosome method(89), and use the I-SceI sticky end for ligation onto complementary single-molecule unzipping constructs. The PHO5 system is usually studied in the context of initiation as opposed to elongation. Nevertheless, the system is ideally suited for us in developing our method, due to the highly positioned nucleosomes with very high occupancy in the repressed state and very low occupancy in the activated state as well as the identified nuclease-hypersensitive sites necessary for our

ligation. In demonstrating the power of our method, we hope to shed light on open questions involving nucleosome shuffling on the PHO5 promoter, and in fact, one group has recently proposed exciting single-molecule experiments different than our own method for addressing this question(87). After validation with well-characterized PHO5 plasmid system, we will move to study of transcription in genomic YLR454 gene, about 8 kilobases long(78, 90) in wild type and mutant strains outlined in Table 1.1. There will be added challenges, notably engineering a unique I-SceI site near the YLR454 gene in each strain and isolation of the genomic chromatin without the benefits of the minichromosome size-based method. Despite these challenges, we view it as a promising avenue, because we are ultimately performing single-molecule analysis, and thus require lesser amounts of lower purity material than many ensemble assays require. Specifically, we may not need to purify the specific genomic fragment before performing the ligation reaction, and will attempt to rely on the I-SceI complementation and the fact that unzipping will only occur when both strands are ligated. In our experience, single-molecule DNA unzipping has such a distinct and sensible signature, that we can rely on this to verify that we have the desired fragment. Below, we extend this idea to our goal of shotgun chromatin mapping, which may preclude the need for this engineered single-site method altogether. We will use the I-SceI / YLR454 system to pursue the experiments outlined in Table 1.

1.4 Shotgun chromatin mapping:

Force (pN)

Force (pN)

Our initial studies will be Solid line is prediction from simple AT / GC model Figure 1.3 Comparison of actual to predicted unzipping force for naked single-site genomic or Open circles are averaged data points DNA. Koch’s OT data (open circles) plasmid chromatin studies, compared with prediction from known due to the desire to carefully sequence and Bockelmann et al. 1997 theory. The correlation value between validate the techniques. these would be high, demonstrating However, engineering the possibility for genome-wide DNA Index (basepairs) shotgun chromatin unzipping. unique genomic restriction DNA Index (basepairs) sites is time consuming and must be carried out in all the mutant strains. Therefore, we will pursue a further innovation we term shotgun chromatin mapping. In this case, the entire genome will be digested with a single restriction enzyme (such as XhoI), creating thousands of known sticky ends. These sticky ends will be ligated onto unzipping constructs and random fragments selected for unzipping. The key is that the actual site in the genome can be determined by the naked DNA unzipping signal. The unzipping force signal is easily predicted from the DNA sequence, dependent on AT versus GC content just as is the case when calculating melting temperatures for PCR primers(80). Thus, we will cross-correlate the actual unzipping data with a library of simulated unzipping signals for all known restrictions sites in the genome. Figure 1.3 shows an example of DNA unzipping data compared with simulation based on known sequence, illustrating the potential power of this method (Koch unpublished data). We currently have LabVIEW software that simulates unzipping for manually cut-and-pasted sequences. During this CAREER proposal, we will develop open-source software for automated access of genome data bases, finding restriction sites, simulating unzipping, and storing the simulated data in publicly accessible data bases. As a precursor to shotgun chromatin mapping, we are also pursuing unzipping of restriction digested genomic DNA (not chromatin). We feel shotgun unzipping of genomic DNA and mapping to specific fragments via the naked DNA unzipping signal is highly likely achievable within a few months. This in and of itself will be interesting for a variety of potentially exciting avenues that could lead to further productive work outside this CAREER proposal. Just a couple examples are: (a) adding back purified proteins to map their binding sites in the genome and relative affinities, (b) mapping lengths of specific regions of DNA, for example, telomeres or highly repetitive regions.

1.5 Potential Risks and Challenges We feel there is a high probability of success because of the expertise of the Osley and Adelman labs and because Koch was co-inventor of the process for mapping and probing protein-DNA interactions by unzipping DNA molecules with optical tweezers. Further, Koch’s method has recently been demonstrated to be able to map the positions of model mononucleosomes to within 3 bp(60). Nevertheless, the proposal is innovative and carries significant risks. We have addressed many of these by the multiple strategies we have outlined above. For example, the minichromosome experiments may present many unforeseen difficulties, which perhaps can be circumvented by pursuing shotgun chromatin mapping in lieu of initial single-site experiments. Another potential complication is that native chromatin may be far “messier” than chromatin arrays reconstituted with purified histones on the positioning sequences. To a large extent, we hope this is true, because this richness in in vivo chromatin structure and post-translational modificiations is the reason we are pursuing these experiments. But interpretation may initially be more challenging than we expect. We have two additional strategies for dealing with this scenario. First, we can adjust the experimental protocol to add washes that will remove most proteins besides the highly stable polymerases and nucleosomes. We can base these methods on existing knowledge of proper treatment of chromatin fibers. Second, as we discuss in the 3rd project below, there is strong potential for high performance computing to assist in interpreting our data. In silico experiments may be able to produce data indicating how the unzipping forces will change for various nucleosome-nucleosome interactions and disruption or addition of histone-DNA contacts by post-translational modifications or remodeling. 1.6 Impact Success in this project will validate a new method for mapping the positions of histones and polymerase on single chromatin molecules isolated from living cells. We will answer specific questions about the epigenetic control of Pol II transcription elongation via histone H2B ubiquitylation and remodeling by FACT, that are currently unanswerable due to limitations of the existing ensemble methods. We expect the unzipping method will be valuable to many other researchers studying cellular processes where chromatin remodeling is essential, including aspects of DNA damage repair which we outline in the next section. Furthermore, isolation of specific chromatin fragments will enable other complementary singlemolecule analyses, for optical analysis of individual chromatin fragments elongated in nanochannels and labeled with antibody-quantum dots with affinity for specific histone epitopes as we describe below in the final research section. If successful, shotgun chromatin mapping (and genomic DNA unzipping) could have impact far beyond the research in this CAREER proposal. Beyond the possibilities already described, shotgun chromatin mapping should be applicable to many other organisms besides yeast, and could allow these methods to make a large impact without need for genetic engineering—for example, in analysis of chromatin from human cell lines or tissues. Project 2: Single-molecule analysis of DNA and chromatin structure in yeast during double-strand break repair 2.1 Background and open questions It has recently become clear that DSB repair shares many of the same chromatin remodeling mechanisms that are used to regulate Pol II transcription. The two major mechanisms of chromatin remodeling include post-translational modification of histones and ATP-dependent disruption or repositioning of histone-DNA contacts in nucleosomes. During DSB repair, both classes of remodeling activities have been observed. The first remodeling event to occur at DSBs involves phosphorylation of the C-terminus of the histone H2A variant, H2AX (-H2AX), which forms a large domain of modified chromatin(93-95). Next, ATP-dependent nucleosome remodeling factors are recruited to DSB sites, where they can lead to nucleosome displacement and eviction.

DNA Strand Resection:

DSBs can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). During repair by both HR and NHEJ, one of the first factors to associate with broken ends is the MRX complex (mammalian MRN), a key DNA damage sensor(96). MRX, in turn recruits the Tel1 kinase (mammalian ATM kinase), which together with the Mec1 kinase (mammalian ATR kinase), phosphorylates H2AX around DSBs. MRN/MRX also plays a role in 5’ to 3’ end-processing, also known as resection(97). By stimulating DNA strand resection, MRN/MRX thus disctates that DSBs will be repaired by the HR pathway. Finally, in yeast, MRX is also required for nucleosome displacement around an HOinduced DSB. Recent data from the Osley lab suggest that this latter role is mediated through the Tel1 kinase, and that the role of MRX in nucleosome displacement is in part due to its control of DNA strand resection (Tsukuda, Osley, unpublished data). Thus, how DNA ends are processed in the context of chromatin, and the role of MRN/MRX in this process, are important questions.

Nucleosome Remodeling: H2A phosphorylation occurs concomitantly with MRN/MRX association with DSBs, but its role is still unknown(98). The H2A C-terminus interacts with DNA near the dyad axis of the nucleosome core and with linker DNA between nucleosomes(99). Phosphorylation could potentially disrupt these interactions, thus leading to an alteration in nucleosome or chromatin structure. An alternate explanation is that -H2AX simply acts as a binding site for chromatin remodeling and DNA repair factors without altering the structure of chromatin. In addition to -H2AX, which is an early remodeling event, chromatin expansion and nucleosome displacement represent later, independent remodeling events at DSBs(100, 101). A key unanswered question is the nature of the initial changes in chromatin structure that occur immediately after formation of a DSB, and the potential role of -H2AX in these changes. These changes are completely unknown because of the absence of sensitive in vivo assays in yeast or higher organisms. We will use single-molecule unzipping (SMU), magnetic tweezers stretching, and tethered particle motion (TPM) analysis of genomic DNA and chromatin to investigate these open questions as outlined in Table 2.1

Table 2.1 Summary of questions that will be investigated regarding chromatin regulation of DSB repair Cell type Selected features seen with New information possibly obtained via singleensemble assays in relation to molecule unzipping (SMU) or tethered particle double-strand break repair motion (TPM) experiments Wild Type Yeast

Rapid H2A phosphorylation Gradual histone eviction from DSB ends Loss of MNase ladder

SMU: establish baseline positions of nucleosomes, nucleosome unzipping structure; examine processivity of nucleosome eviction; correlate left and right side of same single break TPM: establish baseline kinetics of resection; determine processivity of exonuclease.

H2A-S129A in Yeast No S129 phosphorylation

Unable to detect any changes to nucleosome structure by MNase

mre11∆ in Yeast Inactivates MRX complex

Strand resection significantly delayed; histone loss significantly impeded; persistent nucleosome ladder

TPM: Is the delay in resection due to a greatly delayed recruitment or an increased pausing of the endonuclease? Is processivity of exonuclease affected?

arp8∆ in Yeast Defective in INO80 remodeling

Significantly slows histone eviction; persistent nucleosome ladder; resection apparently not delayed

TPM: reconcile the slow histone loss but unchanged resection: does the endonuclease have pronounced pauses at the nucleosome locations?

Histone loss like WT

SMU: Detect subtle changes to nucleosome structure (perhaps loss of particular histone-DNA contacts); detect small changes in histone loss

2.2 Single-molecule unzipping chromatin analysis of DSB repair We plan to use high resolution mapping of nucleosome positions via unzipping of single chromatin fibers. This is the area of highest synergy with project 1 (Pol II transcription elongation), because the biophysical

analysis methods are highly similar (see Fig. 1.1B and replace I-SceI cohesive end with HO cut site overhang). An important note, though, is that we can only use SMU to analyze non-resected DNA, because DNA unzipping inherently requires two intact strands. Please see proposal sections 1.2 and 1.3 above for details on unzipping method and proposed chromatin isolation. Recipient site unzipping analysis: Analysis of chromatin remodeling at the recipient site will be restricted to DSB in cells arrested in G1 by alpha factor which will have no resection. We will pursue analysis of chromatin via the plasmid minichromosome method (with an HO endonuclease cut site) as well as genomic chromatin with the native HO recognition site at the MAT locus. Nucleosome positions will be mapped with high resolution, and questions outlined in Table 2.1 will be addressed. For example, can subtle changes in nucleosome structure be discerned in the presence / absence of phosphorylation on serine 129? Furthermore, as shown in Fig. 1.2B, we anticipate the ability to distinguish between octamers, hexamers, and tetramers and we will investigate whether remodeling after DSB produces these partial nucleosome structures. Donor site unzipping analysis: In contrast to the recipient site, the donor site does not have a break or DNA strand resection. Therefore, we will attempt to analyze chromatin structure at the donor site via SMU and will not be restricted to the G1 cell cycle. Nucleosome positions and structure (octamer, hexamer, tetramer) will be analyzed during defined times after DSB in wild-type and the mutants outlined in Table 2.1 2.3 Tethered particle motion (TPM) and tweezers analysis of resected genomic DNA We will develop methods for analyzing naked DNA isolated from living yeast cells. We will pursue these methods for two reasons. First, isolation of genomic DNA should be much easier than isolation of chromatin, because we can develop methods without worrying about maintaining the integrity of the nucleosomes or polymerase complexes. Second, DNA strand resection is a key feature of DSB repair, and the ssDNA produced by resection is not easily analyzed by the DNA unzipping method (which naturally requires two strands). Our general approach is outlined in Fig. 2.1. A

A

D

Biotinylated hairpin 3’

GAL-HO GAL-HO Not INot I B

B

C

C

Not INot I W

W X

X

3’

3’ 3’

3’ 3’

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Dig-labeled random hexamers Resected Genomic DNA

DNA Ligase + affinity purification

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3’ chromatin 3’ Figure 2.1 (A) Diagram of structure near yeast MAT locus. Nucleosome free region indicated by light circles. (B) Proximal and distal Not I sites will be engineered ~5 kb from HO cut site. (C) A defined time after HO cut, Not I digestion o f genomic DNA will release 5 kb fragments with varying level of resection, but a common 3’ end for affinity purification. (D) Resected fragments ligated to biotinylated hairpins and dig-labeled hexamers. Optional klenow reaction shown by dotted line.

We will induce DSBs at the genomic MAT locus via HO endonuclease(102). After induction and isolation of genomic DNA, the DNA will be digested downstream from the break site at an endogenous nuclease recognition site (Fig. 2.1C). Biotinylated DNA hairpins will be ligated to the known 3’ overhang of the DSB site. Fragments will be isolated via affinity purification with streptavidin microspheres. A digoxigenin label will be added to the 5’ end of resected strand via ligation of dig-labeled random hexamers, and strand will be optionally filled in via a klenow reaction (Fig 2.1D). We will then proceed with single-molecule experiments to essentially measure the distance between the biotin and the dig label, which will mark the beginning and end of DNA resection.

We will use two complementary A B C methods to measure the extent of Force on Magnetic resection on single genomic DNA Bead TPM fragments. The first method (Fig. BiotinPolystyrene streptavidin Bead 2.2 A) will use simple magnetic D Resected Genomic Resected tweezers(31, 103) to stretch single DNA DNA DNA filled-in w/ Klenow tethers in order to remove Dig / anti-dig secondary structure from the ssDNA Coverglass (anti-dig) Coverglass (anti-dig) remaining after strand resection. Figure 2.2 (A, B) Two single-molecule approaches for measuring resection The freely-jointed chain model will kinetics on isolated genomic DNA (see Fig. 2). (A) Amount of ssDNA will be measured via force-extension curve using magnetic tweezers. (B) Length of be fit to the force-extension curve for dsDNA corresponding to resection will be measured via TPM. (C, D) Pooled the tethers in order to extract the results from stochastic simulations of strong pausing of resection caused by (C) number of ssDNA bases in a given positioned nucleosomes and (D) shuffled nucleosomes. Both N=100 molecules, 100 basepair histograms, 90 minutes simulated resection (Koch, unpublished). tether(30). Our current plan is to use magnetic tweezers, due to the expected demand of optical tweezers instrumentation for unzipping experiments, but if higher resolution is required, we can perform the measurements with OT. In the second method (Fig. 2.2 B), the ssDNA will be converted to dsDNA via a klenow reaction. This will remove the secondary structure and tethers will be formed with standard non-magnetic polystyrene beads. The Brownian motion will be analyzed via TPM methods(104-106) and the length of dsDNA will be inferred. Both of these methods should provide the same answers and together will serve as a validation of our data analysis. In Fig. 2.2 C and D, results of simulated single-molecule data are shown, demonstrating hypothetical distinct results from positioned versus remodeled nucleosomes (Koch unpublished stochastic simulations; see research project 3 below). In this hypothetical case, roughly 100 basepair resolution could reveal a difference in resection caused by strongly positioned versus shuffled nucleosomes, in addition to information about the average rate of resection and spread in rates. Alternative based on DNA unzipping To increase likelihood of success, we will also pursue an alternative method for measuring the extent of resection. In this case, we will create unzipping tethers by cutting genomic DNA with an enzyme that cuts roughly 10 kb away from the DSB site, where DNA is expected to remain fully double-stranded. A given single tether will display an unzipping signal predicted by the DNA sequence until reaching the point of DNA resection, at which point the tether will break. Based on the easily modeled DNA unzipping signal, the location of DNA resection will be mapped to within 10’s of basepairs or higher resolution. This method is synergistic with the shotgun chromatin mapping and shotgun DNA mapping discussed in project 1. 2.4 Potential Risks and Challenges The components of this project based on mapping chromatin by unzipping share some of the risks and mitigation strategies described in project 1. For the components based on a analysis of genomic DNA, we feel the likelihood of success is equal to that of the chromatin unzipping. Dealing with genomic DNA instead of chromatin will be much easier, but the biotin / digoxigenin labeling method is yet to be proved and is somewhat more complicated, due to the unknown level of resection. We have planned multiple experimental approaches to mitigate these risks, including complementary tethered particle motion, DNA stretching, and DNA unzipping methods. 2.5 Impact Success in this project will establish new single-molecule methods for analyzing DNA end-processing and chromatin remodeling that occurs in living yeast cells during DSB repair. We will answer important questions regarding the activities of MRX, INO80 and H2A phosphorylation during DSB repair in yeast.

Additionally, DSB repair processes are highly conserved in higher eukaryotes, including mammals. Our methods should be extensible to studies in other organisms, including humans. Project 3: Stochastic simulations, molecular dynamics, and nanochannel analysis The main thrust of our research program in this CAREER proposal is encapsulated in Projects 1 and 2 described above. Success in those projects will certainly serve as a strong foundation for the research career of the PI. There are two additional research components our lab will pursue, which will complement and strengthen the first two projects as well as potentially grow into strong research areas. These are grouped into Project 3 in this proposal due to this nature of supporting the two main research areas. 3.1 Stochastic simulations of polymerase kinetics and other enzymatic processes One of these components is a software application the PI has developed as a tool to aid in interpretation of ChIP assays of RNA Polymerase II transcription. The software application currently has two main components: (a) a stochastic simulation of RNA polymerase transcription in the context of nucleosomes (similar to a recent publication(87)) and (b) a simulated ChIP analysis that processes the simulation Figure 3.1 LabVIEW user interface for setting up stochastic simulation of data. Fig. 3.1 shows a screen shot of transcription. Two steps are shown of a total of 5 in this case. the main user input data for the transcription stochastic simulation. The software allows the user to specify steps similar to what would be done during an investigation of RNA Pol II transcription for a particular gene in yeast. The example shown equilibrates the “gene” in the on state (initiation rate = 0.1 per second) for 700 seconds. Various parameters are set based on literature values or other estimates, for example, the rate of transcription on naked DNA, and likelihood of pausing, rate of termination at a terminator. In the example shown, premature termination is set to essentially zero probability and so is cryptic initiation (feature not yet implemented). In the second step, transcription is shut down by changing initiation rate to 0.005 / s, mimicking the media switch from inducing to repressing. This step is repeated 4 times for 120 seconds (subsequent steps not shown in figure), and positions of polymerases and nucleosomes are captured between each time step (akin to taking aliquots at 120 second intervals in a real yeast Template position experiment). A single run through the experiment is effectively an in silico simulation of a single gene in a single cell. The experiment is then repeated (with different Figure 3.2 Example of capability for displaying animation of individual stochastic results), and results are stochastic polymerase simulations. In this example, transcription was saved for each individual run (a recently repressed. collection of individual cells in

silico). These data—positions of nucleosomes and polymerases at defined times—can then be analyzed by histograms or by feeding into the ChIP simulation application. Another important feature of (a) is the ability to at any time during the simulation “peek” into the simulation and view an animation of the polymerases and nucleosome kinetics. Figure 3.2 shows an example of the animation during a step after the “gene” has been shut down, showing the polymerases clearing off the template to the right as nucleosomes reassemble to the left. Effectively, we are catching a glimpse of the “wave” of Pol II as it travels down the gene as seen in real ChIP data for the YLR454 gene(90). This ability alone can lend great insight to complicated scenarios—for example, if a nucleosome remodeling defect has been implemented or if non-specific termination is significant. The snapshots taken from the simulation (akin to aliquots of yeast cells taken at time intervals) can then be fed into the ChIP simulation software. This software application takes the individual simulation results, and randomly segments the DNA molecules into fragments according to user specified shearing parameters (e.g. 600 bp average length, 100 bp standard deviation). The fragments are then sorted according to the presence or absence of a polymerase or nucleosome and the resulting pieces are then “detected” by user-specified primer pair locations. These “detection levels” can then be plotted as would real-time PCR results in actual ChIP assays. An example of simulated YLR454 ChIP data is shown in the figure, paralleling the real ChIP data from Mason and Struhl(90). 3.1.1 Research plan for kinetic simulation We currently have a working platform that is producing valuable results. Key things that will be done under this CAREER research are:  

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Improving the speed of the code Validating results of actual yeast ChIP results for YL454 transcription, and attempting to validate a prediction for a new experiment. Open Science: Publishing of the code and providing full access to all of the code via OpenWetWare and SourceForge. Creation of a more user-friendly toplevel application plus installer & documentation that users can download and use to simulate their own scenarios. To be available via OpenWetWare.

Promoter 5’ ORF ORF ORF 3’ ORF

Time (s) Figure 3.3 Example of simulated ChIP for a stochastic simulation of transition from activated to repressed transcription. Polymerases are seen leaving the 5’ end of the gene before 3’ end, as expected.

3.2 In silico unzipping experiments. We anticipate that chromatin mapping by unzipping will be a very powerful technique. However, it’s reasonable to expect that detailed information about individual post-translational modifications or other nucleosome structural modifications may not be easily discernible in the unzipping data. Two strategies are being pursued to augment the unzipping technique with this capability: in silico molecular dynamics simulations of unzipping to elucidate finer detailed information about unzipping forces, and single-fiber imaging with specific antibodies (in the next section). The increased forces observed when unzipping through protein-DNA complexes arise from collections of strong protein-DNA interactions. Elimination of some of these interactions can have a dramatic effect on the unzipping signature (see, e.g., the difference between tetrasome and nucleosome in Fig. 1.2 B). It is often thought that post-translational modifications modulate the strength of the histone-DNA interaction, and this has been seen in some single-molecule

experiments (37). Thus, we anticipate that the unzipping signature may differ depending on the PTMs of a given histone (e.g. H2Bub or -H2AX). Predicting the force changes is a daunting task, as is calibrating each potential modification (and all of the combinations) on in vitro reconstituted systems. However, computing power continues to increase, and we are now at the point where it is within reach to consider molecular dynamics simulations of the unzipping of large protein-DNA complexes(107). The PI’s lab and Dr. Chris Lorenz’s lab of King’s College London submitted a letter of intent for a Human Frontier Science Program young investigator award in 2008 (score was in the 15th to 30th percentile) and are planning on submitting a revision in 2009 to fund a project in pursuit of developing the capability of unzipping nucleosomes and Pol II-DNA complexes in silico. The project will require significant effort and is outside the scope of this CAREER award. However, as part of the CAREER proposal, we want to initiate this project and begin generating preliminary data towards our end goal. Specifically, we already have in hand a significant amount of unzipping data for naked DNA and model protein-DNA systems (such as EcoRI). We want to use this data to compare with test cases for the steered molecular dynamics simulations. Conversely, once the model is working for naked DNA and model protein-DNA systems, we will make predictions for new model systems and attempt to validate them with new unzipping data. This preliminary data will greatly aid in obtaining independent funding, and could lead to a very powerful enhancement of our chromatin mapping technique—the ability to discern single epigenetic marks on single chromatin fibers. 3.3 Imaging-based single-molecule chromatin mapping ChIP currently has a key capability of using antibody specificity for post-translational modifications of b histones; histone-variant specificity; and polymerase CTD phosphorylation-state specificity. Thus, a variety of antibodies are available for these specific epitopes. In this research component, we plan to leverage these Figure 3.4 (a) 130 nm x 400 nm channels with porous-walls antibodies, in combination with our site-specific formed from silica nanoparticles. (b) confocal microscopy of chromatin isolation work and high resolution imaging to lambda DNA molecules in the channels—loaded solely via capillary action (no pumps or electrical driving force). complement our single-molecule unzipping work. Chromatin isolation will be similar to project 1 above, but with possible routine use of formaldehyde crosslinking. Antibodies will be coupled to gold nanoparticles or quantum dots, and the chromatin labeled with these nanoparticle-conjugated antibodies. The key innovation here will be the elongation of the chromatin via porous-walled nanochannels that we have been working with(108) (see Fig. 3.4). In these devices, DNA molecules can be quickly elongated by simply placing a drop of DNA-containing solution in the well. We will attempt the similar experiment using chromatin labeled with gold or QDs. The expected spacing between nucleosomes will be much less than the standard optical resolution limit, and thus we will analyze the elongated molecules using either super-resolution optical microscopy (in collaboration with Brueck laboratory), or electron microscopy (for the gold nanoparticles). 4. Educational Integration and Broader Impacts Our lab believes strongly in our mission of advancing molecular cell biology, and therefore our goals are well aligned with those of the NSF for integrating research with education and maximizing the broader impacts of our research. Broadly communicating our results and methods, teaching at all levels, and training future leaders in biophysics research together will greatly multiply the impact of our research discoveries. Below, we describe our plans towards this goal in three distinct areas: open science, innovation in undergraduate education, and community outreach.

One way of leveraging our impact is in recruitment and training of underrepresented minorities to biophysics, and this is an underlying component of all three of our goals below. We are at an advantage in this area, because of the unique demographics of the University of New Mexico and the surrounding area. UNM is designated a Hispanic-Serving Institution by the US Dept. Ed (http://www.ed.gov/about/offices/list/ocr/edlite-minorityinst.html) and other government agencies. Additionally, the local and state population consists of a high proportion of minorities underrepresented in science. Therefore, we plan on passively leveraging this advantage by the fact that our programs are carried out in this environment. We already have had one success story via our participation in the UNM PREP program, an NIH-funded program that funds post-baccalaureate minority students for a year of research and study at UNM as a stepping stone for graduate school in the biological sciences. We were fortunate to host a successful fellow this past year, Diego Ramallo Pardo, who will be attending Stanford for graduate work in biophysics this Fall and hopefully continuing to collaborate with our lab. 4.1 Open Science Since early 2007, our lab has been actively participating in OpenWetWare (OWW), an Open Science site hosted by MIT (http://www.openwetware.org/wiki/Koch_Lab). The goal of open science is complete open sharing of all data and knowledge generated in the laboratory and licensing of this information with a very permissive Creative Commons license allowing derivative works by other laboratories. While we are striving towards this goal, our lab has not currently made the leap to full openness, for two main reasons: the need to work out a collective plan with all of our collaborators, and the perceived additional risk to young graduate students who have already assumed the risk of working for a young untenured PI. However, we have been taking many steps in the right direction. These include: publishing on OWW several detailed single-molecule protocols; publishing on OWW (with co-author permission) two working drafts of papers related to postdoctoral work and graduate work; posting of a software application for versatile feedback control of optical tweezers; and hosting courses on OWW, one of which we will describe in section 4.2. We will evaluate our success in this goal by our progress towards complete openness, the page hits and other interest in our work that we can measure, and the number of students and collaborators we recruit to OWW who continue to use OWW after moving on from our lab. Our ability to succeed in our open science goals will be greatly aided by the CAREER award and the stability and assurance it will provide our lab. 4.2 Integration of research with undergraduate education Dr. Koch teaches two courses so far in the Dept. of Physics and Astronomy: Physics 102, “Introduction to Physics,” and Physics 307L, “Junior Lab.” Physics 102 is a course intended for non-science majors with a minimum of mathematics. The students enrolled in this course cover a broad spectrum and include many of the future lawmakers and other science funding decision makers. So, one of Dr. Koch’s goals in teaching the course is for the students to come away from the course sharing in the excitement and understanding the concepts and methodology of physics and to strengthen future support of science. Our biophysics research is integrated into the course in two ways: first, many of our research projects fit very well with topics in the course: for example, green fluorescent protein and fluorescence microscopy are explained during the explanation of atomic physics, electron energy levels, and electromagnetic radiation. Second, physics graduate students are involved in the teaching of the course. During the Spring 2008 course, the TA for the course and two of the graduate students in Koch Lab prepared, rehearsed, and carried out one 75 minute lecture. Junior Lab is the first “real” physics lab course that physics majors at UNM enroll in. The current lab modules are common modern physics experiments, including speed of light measurement, radioactive decay, millikan oil drop, planck’s constant, etc. The main innovation with this course is that Dr. Koch hosts this course completely as Open Science, on OpenWetWare (http://www.openwetware.org/wiki/Physics307L), with the only exception being communications that must

be kept private. The students keep all of their notes electronically on OpenWetWare, along with their lab write-ups and formal reports. Additionally, all of the written feedback from Dr. Koch (with the exception of letter grades) is also carried out in public on OWW. The results of the first trial of this Open Science experiment were very rewarding: of the 16 students, 4 of them continued to use OWW subsequent to Junior Lab in their own coursework and lab work. Furthermore, the PI feels that there are significant benefits to exposing and training students in open science at this early stage in their research careers and will ultimately make a large impact on the next generation of open scientists. In addition to the open science component to Junior Lab, we intend to add new biophysics modules to the course as part of this CAREER award. We have budgeted a small amount of money for equipment needed to add these modules, which are specifically: tethered particle motion (TPM) analysis of single DNA molecules (which we have already added to Dr. Lidke’s undergraduate / graduate biophysics course), optical tweezers / magnetic tweezers experiments, and PDMS microfluidics. Additionally, we have commitment from the chair of UNM Physics Dept. to support our goals of adding biophysics experiment modules with college and department funds when needed. 4.3 Community and school outreach in Albuquerque, NM Dr. Koch has participated in community scientific outreach since early in his graduate career and it remains and enjoyable and valued activity for him. During Dr. Koch’s first two years at UNM, he has judged the Central New Mexico Science and Engineering Challenge (Middle School Microbiology) and the Cleveland Middle School Science Fair. Additionally, our lab has hosted a group of 24 science students from Menaul Middle School for an afternoon of hands-on science demos and tour of the nanofabrication facility. Dr. Koch has also given a presentation on nanomanipulation in the biological sciences to local Middle and High School science teachers at the NNIN-sponsored summer “nanocamp.” Our plan for community outreach as part of this CAREER proposal is two pronged: to continue and grow the outreach efforts of the PI; and to continue to encourage, value, and reward community outreach by the graduate, undergraduate, and postdoctoral members of our lab. In addition to continuing the science fair judging and middle school inreach activities, we plan on expanding our relationship with the local middle school science teachers we have been working with, Mr. Victor Chacon (Cleveland Middle School) and Mr. Alex Cimino-Hurt (Menaul Middle School). While we have not yet made plans, possible ideas are to work with Mr. Chacon’s Math, Engineering, Science Achievement (MESA, http://www.nmmesa.org/) program and involvement of science teachers and students in summer laboratory research projects.

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Biographical Sketch Steven J. Koch Position / Affiliation:

Assistant Professor Department of Physics and Astronomy Center for High Technology Materials University of New Mexico Albuquerque, NM 87106

(a) Professional Preparation University of Michigan Ann Arbor, MI

Physics (honors)

B.S. 1996

Cornell University Ithaca, NY

Physics (biophysics minor)

M.S. 2000 Ph.D. 2003

Sandia National Laboratories and Center for Integrated Nanotechnology Albuquerque, NM

Biophysics and Nanotechnology

2003 - 2006

(b) Appointments 2006 -

Assistant Professor, Dept. of Physics and Astronomy and Center for High Technology Materials

2004 - 2006

Postdoctoral Fellow, Center for Integrated Nanotechnologies and Sandia National Laboratories

2003 - 2004

Postdoctoral Appointee, Sandia National Laboratories

1997 - 2003

Research Assistant, Department of Physics, Cornell University

1996 - 1997

Teaching Assistant, Department of Physics, Cornell University

(c) Publications (i) Most relevant to this proposal Koch SJ, Shundrovsky A, Jantzen BC, and Wang MD (2002). "Probing protein-DNA interactions by unzipping a single DNA double helix." Biophys J 83(2): 1098-105. Koch SJ, and Wang MD (2003). "Dynamic force spectroscopy of protein-DNA interactions by unzipping DNA." Phys Rev Lett 91(2): 028103. Koch SJ, Thayer GE, Corwin AD, and de Boer MP (2006). "Micromachined piconewton force sensor for biophysics investigations." Applied Physics Letters 89(17): 173901-3.

Xia D, Gamble TC, Mendoza EA, Koch SJ, He X, Lopez GP, and Brueck SR (2008). "DNA transport in hierarchically-structured colloidal-nanoparticle porous-wall nanochannels." Nano Lett 8(6): 1610-8. Yeh RC, and Koch SJ (2008). "Versatile Control System for Automated Single-Molecule Optical Tweezers Investigations " Non-peer-reviewed open-source software. URLs: http://openwetware.org/wiki/Koch_Lab:Publications/Drafts/Versatile_Feedback http://sourceforge.net/projects/tweezerscontrol/ (ii) Other significant publications Rivera SB, Koch SJ, Bauer JM, Edwards JM, and Bachand GD (2007). "Temperature dependent properties of a kinesin-3 motor protein from Thermomyces lanuginosus." Fungal Genet Biol 44(11): 1170-9. Liu H, Spoerke ED, Bachand M, Koch SJ, Bunker BC, and Bachand GD (2008). "Biomolecular Motor-Powered Self-Assembly of Dissipative Nanocomposite Rings." Advanced Materials in press. (d) Synergistic Activities 1. Innovations in teaching: Open Science for Junior Physics Laboratory. Student work and instructor feedback carried out in public on OpenWetWare wiki site (http://www.openwetware.org/wiki/Physics307L). 2. Recruitment of underrepresented groups: 2007-2008, Mentor for fellow in UNM’s NIH-funded PREP program for underrepresented minorities. 3. Service to science and engineering community (Albuquerque): 2004-present, Middle school science fair judging in Albuquerque; 2007-present, Middle school outreach (e) Collaborators & Other Affiliations Collaborators and Co-Editors Bachand, George D. (Sandia National Labs); Bachand, Marlene (Sandia National Labs); Bauer, Joseph; de Boer, Maarten P. (Carnegie Mellon U.); Corwin, Alex D. (General Electric); Edwards, John (Johns Hopkins); Gamble, Thomas (U. New Mexico); Liu, Haiqing (Sandia National Labs); Lopez, Gabriel P. (U. New Mexico); Mendoza, Edgar (Redondo Optics); Rivera, Susan B. (Sandia National Labs); Spoerke, Erik D. (Sandia National Labs); Thayer, Gayle E. (Sandia National Labs); Xia, Deying (U. New Mexico); Xian, He (U. New Mexico); Yeh, Richard C. (Madoff Investment Securities)

Graduate Advisor and Postdoctoral Sponsor Michelle D. Wang (Cornell U., PhD Advisor) George D. Bachand (Sandia National Labs, Postdoc Advisor)

Thesis Advisor Lawrence J. Herskowitz (U. New Mexico, current advisee), Anthony S. Salvagno (U. New Mexico, current advisee), Douglas A. Read (U. New Mexico, current minor advisee)

Facilities, Equipment, and Other Resources The group of Dr. Koch maintains an approximately 500 square foot laboratory at the Center for High Technology Materials (CHTM) for experimental biophysics research equipped with the following: 

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An optical trapping setup based on an Olympus IX-71 inverted research microscope with bright field and epi-fluorescence capabilities and PLANSAPO 100x NA 1.4 oil immersion objective. Andor Luca electron-multiplying CCD (EMCCD) camera. Blue Sky Research 390 milliwatt 1062 nm laser diode (long-term loan from Keir Neuman, NIH). Laser line optics (Newport), dichroics (Chroma). Phresh Photonics quadrant photodiode with integrated current amplifier. Currently being researched and acquired with existing funds: piezo translation stage or piezo mirror deflector (Mad City Labs). Equipment is mounted on a 3 foot x 4 foot TMC optical table. Within a year, the equipment will be moved to a new additional space we have acquired in the CHTM, with approximately 200 square feet and a 4 foot by 6 foot optical table with Newport pneumatic vibration isolation system. Optical tweezers control hardware and software. Dell Optiplex 755 minitower with 4 GB RAM, 2x250 GB RAID-1 fault tolerant hard drive, dual-monitors. CHTM site license for complete LabVIEW development suite, including NI-Vision image acquisition and processing libraries. Custom optical tweezers control software (written with Richard Yeh, now an open source project: http://openwetware.org/wiki/Koch_Lab:Publications/Drafts/Versatile_Feedback)-software is currently functioning in LabVIEW 6.1 and we will soon port to LabVIEW 9. National Instruments M-series multifunction DAQ with BNC breakout. Lab computing network. Dell PowerEdge 840 running Windows Server 2003, Exchange Server 2003, and IIS. 4x500 GB RAID-5 fault tolerant hard drive configuration. 1 GB RAM. Linksys RVS4000 Firewall with Virtual Private Networking (VPN)—provides secure access to lab domain including Windows remote desktop. APC Uninterruptable power supply. Four Dell Optiplex desktop stations for routine use on lab domain (acquired via UNM surplus). Two Compaq workstation laptops for on demand data acquisition, data analysis, and electronic lab notebook usage. Equipment for basic molecular biology and sample preparation: Positive pressure sterile hood with UV sterilizer; Thermo Sprint PCR Thermocycler; Tuttanauer-Brinkman autoclave; Ohaus analytical balance; micro centrifuge; Fisher Isotemp oven; bath sonicator; vortexer; hot plate stirrers; microwave; -20C non-frost-free freezer; +4C refrigerator; two sets of eppendorf pipetmen; routine glass and plasticware.

Additionally, we have access to the following resources in support of the CAREER proposal: 

No-cost unlimited access to OpenWetWare (OWW) public open science wiki site (http://www.openwetware.org/wiki/Koch_Lab). This site is used for our public web site, where we share protocols, some publications in preparation, and also host our Junior Lab course (http://www.openwetware.org/wiki/Physics307L). Additionally, OWW

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provides our lab with a hosted private wiki at no cost and unlimited access. Both wiki sites are powered by MediaWiki, the same software that powers Wikipedia. Our private wiki is used for all of our day to day electronic lab notebooks and lab communication, and because of the common software platform, facilitates the easy and timely transfer of information to the public website (see Lorrie LeJeune letter). CHTM-subsidized access (approximately $300 per user per month) to the state-of-theart cleanroom and laboratory facilities at the CHTM (an NSF NNIN user facility), including advanced materials growth and characterization, semiconductor processing, and nanostructures fabrication. Notable facilities that may be used in support of the proposed research are equipment for fabrication of polymer- (PDMS) based microfluidics system (including mask creation, spin-coater, UV photolithography, developers, and plasma cleaner), scanning and transmission electron microscopy (SEM and TEM), confocal microscopy, and imaging interferometric lithography. These facilities are used by our collaborator’s group as well (see Dr. Brueck support letter). UNM Center for High Performance Computing (CHPC). The CHPC supercomputing facility is located on the main campus of the University of New Mexico. It operates several large supercomputers, including the 32 node/128-processor nano Linux cluster (Intel Xeon EM64T architecture; 4 cores/node, 2 GB RAM/core; 160 GB disk/node; Myrinet-2000 interconnect), the 16-processor shared memory poblano machine (IBM 64bit Power5 architecture; 256 GB RAM; AIX OS), and the new 2.2 TFlop SGI Altix ICE 8200 machine (22 nodes, Xeon quad core, InfiniBand), which is part of the New Mexico Computing Applications Center (NMCAC). Because of Dr. Koch’s membership in the management committee of the NSF-funded Nanoscience and Microsystems (NSMS) IGERT program at UNM as well as his involvement in Nanoscience, his group is afforded high-priority access to these machines (see Dr. Caudell and Dr. Atlas letters). Biological work with yeast will be carried out in the molecular genetics laboratory of Dr. Mary Ann Osley in the UNM Dept. Molecular Genetics and Microbiology, School of Medicine. Necessary equipment and materials for yeast growth, molecular biology, chromatin isolation, DNA isolation, Southern blots, etc. will be provided by Dr. Osley. Specialized materials, such as non-routine enzymes (e.g. I-SceI, lyticase) and biotinand dig-labeled nucleotides will be provided by Dr. Koch’s laboratory under CAREER funding. (See Dr. Osley letter.) Stalled RNA Polymerase II unzipping constructs will be provided by Dr. Karen Adelman of the National Institute of Environmental Health Sciences (NIEHS) of the NIH. (See Adelman letter.)