Bio2000 Re Combination

Basic models of HR (based on how the HR is initiated) • •

Holliday model: 1 single strand break on each paired DNA double helix Meselson-Radding model: a single strand break on only one of the paired DNA double helix, then anther single strand break on the other strand

•

Double strand break model (more plausible!!!)

• HR can occur in germ cells during meiosis as a controlled process to produce diversified off-springs of an organism • HR can be used to switch mating type in yeast • HR can occur in somatic cells as a DNA repair mechanism • HR is used to resolve stalled replication forks

Initiation of Homologous Recombination by Single Strand Break Holliday Model

Genomes, Figure 14.29

Meselson-Radding Model

Double strand break induced recombination.

Genomes, 2nd ed. Fig14.33

Proteins may be required for involved in HR •

Initiation of DNA strand breaks: nuclease in some cases

•

Helicase and end processing enzymes (nuclease)

•

Single strand binding proteins (SSB or RPA)

•

Strand invasion protein: Rec A in E. Coli and its family members in eukaryotes

•

Proteins helping RecA family to load on single strand DNA and displace RPA

•

Ligase to form Holliday junctions

•

Helicase in branch migration

•

Nuclease to resolve holiday junctions

•

Others: DNA polymerase, topoisomerase, chromatin remodeling

Proteins involved in DSB-induced HR E. Coli

Yeast

Mammalian

DSB

Damage replication

HO, Spo11, Damage Stalled replication forks, etc

Nuclease, Damage, Stalled replication forks, etc

End processing

RecBCD

MRE11/RAD50/XRS2 ?

MRE11/RAD50/NSB1 ??

Single strand binding protein

SSB

RPA

RPA

Strand invasion

RecA

RAD51 DCM1

RAD51, RAD55, RAD57, BRCA2?

RAD52 RAD54 ?????

RAD52 ?, RAD54?, BRCA2? ?????

?

?

Mus8?

Mus8?

Helper for RAD51’s & facilitators for strand annealing, etc Branch migration

RuvAB

Resolution of Holliday RuvC J.

Proteins involved in mammalian HR

End processing: MRN nuclease complex: MRE11-RAD50NBS1 Other nucleases Recombination: RAD52 RAD51 RAD54 (helicase) XRCC3 BRCA2 RPA DNA polymerase, PCNA, Other Helicase: WRN: Werner’s syndrome gene BLM: bloom syndrome gene Accessory proteins (??): Fanconi Anemia Proteins BRCA1 ATM ATR

Ratio and dynamics of these proteins are important !!! Valerie K & Povirk LF, Oncogene, 2003.

Rec A protein and strand invasion

RAD51 protein family • E Coli: RecA • Yeast (Saccharomyces cerevisiae): RAD51, RAD55,RAD56, DCM1 • Mammalian cells: RAD51, RAD51B, RAD51C, RAD51D, RAD55, RAD57, XRCC3 Other proteins that may facilitate the strand invasion or annealing: RAD52, BRCA2(?), etc.

Double-Strand Break Repair Homologous Recombination (HR) Binding and processing of the ends to reveal single stranded 3’-ends

RAD51 RAD52 RAD54 XRCC3 Helicases RPA BRCA1 BRCA2 etc.

(major)

Single stranded DNA invades the homologous template DNA synthesis and branch migration to form a complicated structure named Holliday Junction (HJ)

(minior)

Resolving of HJ to complete the repair process

Introduction: DNA Repair and Carcinogenesis

Some interesting observation but full mechanisms are not so clear •

Mismatches often slow down or inhibit HR

•

In mismatch repair deficient cells, higher HR is often seen

•

HR confers hypersensitivity to cross-links

•

Activities of HR is often higher in S and G2 phase

•

Resolution of replication blockage is often coupled with HR

•

Homologous recombination between two similar (but not completely homologous region is sometimes called homeologous recombination.

Single Strand Annealing Single-strand annealing - a repair pathway shared between NHEJ and HRR. SSA could occur when, at a DSB, the DNA strand is resected by a nuclease, for example, MRE11 to leave ssDNA overhangs. The length of the overhangs and the extent of homology ranging from microhomologies to several hundred bases or longer most likely determine how SSA is executed. In the case of long homologies between direct repeats on the overhangs, RPA and RAD52 are necessary for facilitating DNA pairing followed by removal of the tails by ERCC1/XPF nuclease and gap filling by DNA polymerase as shown in the figure. Sensors, such as ATM, may signal and attract nucleases to the DSB. Microhomology-based SSA, although similar in principle, probably has quite a different enzymology

Valerie K & Povirk LF, Oncogene, 2003. 22:5792

Double-Strand Break Repair Non-homologous End-Joining (NHEJ) 5' 3'

Steps: (Exonuclease)

5' 3'

(Anneal)

5' 3'

2. DNA ends processed Reveal “microhomologies” at ends 3. Micro-homologies alignment 4. Ligase seals

(Synthesis and ligation)

5' 3'

•Imprecise (deletions and small insertions) •Key role in chromosomal translocations •Important in V(D)J recombination

Outline of the two principal pathways of DNA double-strand break repair. NHEJ involves six known proteins, in a coordinated sequence to ensure ligation of the broken ends. Loss of DNA sequence occurs at the site of repair. HR involves multiple proteins in a complex to allow strand exchange and resolution. The process of HR can be an error-free process. Simon N Powell and Lisa A Kachnic. 22: 5784-5791