Lecture Outline 05

DNA Replication, Repair, and Recombination (3Rs): Enzymes, Reactions, and Their Deficiency in Genetic Diseases (Bio2000 Syllabus – Fall 2005)

Binghui Shen, Ph.D. Professor and Director Department of Radiation Biology City of Hope National Medical Center and Beckman Research Institute 1500 East Duarte Road Duarte, CA 91010 Tel: (626) 301-8879; Fax: (626) 301-8280 Email: [email protected] http://bricoh.coh.org/shenlab

1

I. DNA Replication Ultimate goal of DNA replication: to duplicate the genome precisely DNA structure review Bacterial genome: o Naked o Circular (no end) o Relatively short o No cell cycle specific duplication Eukaryotic genome: o Embedded with many proteins o Organized in as structure called chromatin, nucleasome o Have multiple copy of the linear chromosomes o The end of the chromosome is called telomeres o Much longer than prokaryotic cells o Duplication is cell cycle specific Semi-conservative DNA replication o Classical experiment leading to this conclusion: Meselson-Stahl experiment): (reading: section I3.I.I. page 385-388, figure 13.3 on page 387) o The simple model on linear DNA (slide figure) Other replication modes: (reading: I3.1, Figure 13.6)) o Displacement (Figure/slide) o Rolling circle (Figure/slide) Initiation sites recognition and initiation complex (reading: I3.2.1, p391) Bacterial initiation process (Reading: Figure 13.8) Yeast initiation process (Figure 13.9) Mammalian initiation process: 5’-3’ unidirectional DNA synthesis by DNA polymerases Basic activities of DNA polymerases: a. All DNA polymerases have the 5’-3’ polymerase: use the 3-OH group for a nucleophilic attack on the interior phosphorus atom. Therefore, it needs the free 3-OH group b. 3’-5’ exonuclease c. 5’-3’ exonuclease d. What are nuclease, exonuclease? Okazaki fragments on the lagging strand E Coli: 1000-2000 nt, need 4000 priming event Eukaryotes: ~200 nt,

2

Partial list of DNA polymerase 5-3’ 5’-3’ polymeras exonucleas e e Type I (E.coli) Yes Yes Type II (E.coli) Yes No Type III (E.coli) α

Yes

No

3’-5’ Function exonucleas e Yes Repair Yes Repair (minor form) Yes Replication

Yes

No

No

β δ ε

Yes Yes Yes

No No

Yes Yes

γ

yes

No

Yes

Replication Coupled with delta Priming (~20nt) Repair Replication Detection of DNA damage in replication Mitochondrial replication

Composition Single peptide Single peptide Multiple peptide 4 subunit

2-3 subunits > 1 subunit 2 subunit

Synchronized view of replication forks and key proteins involved (view pictures) o Helicase o Hexameric ring o ATPase o ATP dependent activity o Attached to the lagging strand o Experimental assays to measure helicase activity o RNA primerase: o Tightly associated with DNA Pol-α o Synthesize 8-12 nt of RNA o Then hand over to DNA Pol-α o In bacteria, 4-15 nt of RNA, then hand over to DNA Pol-III directly o Replication Protein A (RPA): single strand DNA binding protein o Clamp loading protein: RFC o Proliferating Cell Nuclear Antigen (PCNA): o Removal of RNA primer: Fen-1 and/or RNase H 3

Replication - Topological problem The topological problems 1. In DNA replication (show figure) 2. After the DNA is replicated, the tangling of double stranded DNA 3. During transcription 4. During cell division: tangled DNA need to be un-tangled 5. DNA repair process Anything involved in DNA reorganization may encounter the topological problem. Steps in topoisomerase actions and classification: 1. DNA binding 2. DNA cleavage 3. Strand passage 4. DNA re ligation 5. ATPase is required for Topo-II in order to turn over the enzyme, but not required for topo-I Topoisomerase and classification a. Type I vs. Type II b. Type IA vs. Type IB: based on the activity Type IA (link to 5’ –phosphate) Type IB (link to 3’ –phosphate) c. Type IIA vs. Type IIB: based on structure of the protein d. A partial list of topoisomoerase (show table in Annu Rev. Biochem, 2001. 70:369) Topo Inhibitors: Covalent/cleavage complex stabilizer (topo poison): • Dependent on DNA replication, mitosis, transcription, and other DNA metabolizing activity. • Can deprive the cell of active Topos, thus prevent normal process (if the enzyme is essential), or act as DNA damage. • However, in yeast Topo I is not essential, depriving topo I will not kill the cell but can generate DNA damage • Examples: o Camptothecins (topo I): topotecan, irinotecan (CPT-11) o Epipodophyllotoxins (Topo II): etoposide, teniposide o Aminoacridines (topo II): amasacrine o Anthracyclines (topo II): doxorubicin Catalytic inhibitor (topo suppressor): all other types 4

5

The end problem during DNA replication – telomeres 1. The structure of eukaryotic telomeres 2. Where does the shortening come from? 3. The mechanisms to add the missing part back 4. Telomerase structure and function 5. Relevance to cancer

6

II. DNA repair 1. 2. 3. 4. 5.

Photo-reactivation Mismatch Repair Base excision repair Nucleotide excision repair DNA double strand break repair

7

III. DNA Recombination 1. Types of recombination a. Homology directed recombination, or homologous recombination b. Site specific recombination c. Transposition and retro-transposition 2. What is DNA homologous recombination? a. Early view by geneticists (during meiosis): Linkage: genes on the same chromosome should travel together during meiosis. Recombination: genes on the same chromosome can be separated during meiosis b. One chromosome level: gene conversion and crossover c. On molecular level: a process involves cut and relegation of DNA 3. Basic models of HR (based on how the HR is initiated) a. Holliday model: 2 single strand break b. Meselson-Radding model: 1 single strand break model, then another single strand cut c. Double strand break model: 4. Several technical terms: a. Crossover: exchange of homologous DNA segments between two homologous chromosomes b. Gene conversion: conversion of a DNA segment from a chromosome to its homologous chromosome c. Synapsis: the pairing process of homologous sequences d. Strand invasion e. Strand exchange f. Heteroduplex g. Holliday Junction h. Branch migration 5.

The RecBCD/Chi pathway in E. Coli recombination a. RecBCD protein complex in bacteria: b. Contains an endonuclease activity that makes single strand break in DNA c. DNA helicase activity that unwinds the complementary strands in DNA d. Initiate the HR process in bacteria.

6.

Other proteins involved in HR a. RecA/RAD51 protein family (RAD51, RAD55, RAD57): strand invasion b. BRCA2 c. Rad52, RAD54

8

7. Roles of DNA recombination in DNA double strand break repair 8. Roles of DNA recombination in restart stalled replication forks 9. Roles of HR in meiosis 10. Roles of HR in mating type switch 11. Mismatches and HR: mismatches inhibit HR 12. Application of homologous recombination in medical research: gene “knockout” 13. Technique to measure recombination in mammalian cells a. Integration experiment b. DSB induced HR on artificially installed substrate 14.

Defects of homologous recombination and cancer a. BRCA2 and BRCA1 gene and hereditary breast cancer and other cancer b. Fanconi’s Anemia c. Hyper-sensitive to DNA damage induced by cross-link reagent, such as mitomycin C, cisplatin d. Moderate sensitive to ionizing radiation in mammalian, but hypersensitive in yeast e. Chromosome translocation, FISH analysis f. Others: replication checkpoint activation

15. Examples of site specific recombination: a. Serine-type recombinase: tetramer, 4 cuts and then switch the ends b. Tyrosine-type recombinase: 2 cuts, then holiday junction, then 2 more cuts to resolve HJ c. Directionality of site-specific recombinase d. The Cre recombinase and its application in conditional gene knockout. e. Integration of λ-DNA and bacteria DNA f. V(D)J recombination in immune system 16. Transposition and retro-transposition: a. Cut and patch transposition b. Replicative transposition

9

IV. Paper for Discussion Wang Y. et al., (2005) Mutation in Rpa 1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice. Nature Genetics, 37, 750-755

10