Recombinational Repair

Andrew PierceGraduate Center for Toxicology

University of Kentucky

TOX 680

Recombination: bringing DNA strands together in new ways

Spontaneous orRad52 mediatedStrand annealing

RecA orRad51 mediated

Strand invasion

Recombination is used to repair topological damage

Single-strand gap repair

Double-strand break repair

Recombination is used to repair topological damage

Replication across a nicked template

Ends need to invade the duplex

Template Switching

Dale B. Wigley, Clare Hall Laboratories, UK

•Replication fork with blocked leading strand branch migrates backwards and/or newly synthesized leading strand invades to pair with the newly synthesized lagging strand•Leading strand is polymerized using the newly synthesized lagging strand as a template•Resulting Holliday junction branch migrates back to reconstitute active replication fork

Rad51

• Eukaryotic homolog of E. coli RecA • Binds single-stranded DNA and double-stranded DNA• Searches for regions of homology• Exchanges homologous strands

Image is from the cover of the March 26, 1993 issue of Science

Homology search mechanism

Flip the puckering of the ribose ring?

PNAS Vol. 95, Issue 19, 11071-11076, September 15, 1998Taro Nishinaka, Akira Shinohara, Yutaka Ito, Shigeyuki Yokoyama, and Takehiko Shibata

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Rad51 is required for cellular viability

The EMBO Journal (1998) 17, 598–608 Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell deathEiichiro Sonoda, Masao S. Sasaki, Jean-Marie Buerstedde, Olga Bezzubova, Akira Shinohara,

Hideyuki Ogawa, Minoru Takata, Yuko Yamaguchi-Iwai and Shunichi Takeda

4 Gy

Rad51-

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The EMBO Journal (1998) 17, 598–608 Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell deathEiichiro Sonoda, Masao S. Sasaki, Jean-Marie Buerstedde, Olga Bezzubova, Akira Shinohara,

Hideyuki Ogawa, Minoru Takata, Yuko Yamaguchi-Iwai and Shunichi Takeda

Loss of Rad51 results in chromosome fragmentation

A:B:C-D:E:

Untreated4 Gy (+3h)4 Gy (+8h)Rad51- (+21h)

Rad51 paralog family

Rad51: ATP-hydrolysis-mediated strand exchange

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Nature Reviews Cancer 2; 351-360 (2002)ONCOGENIC TYROSINE KINASES AND THE DNA-DAMAGE RESPONSE

DMC1: meiotic paralog

XRCC3-deficient irs1SF cell line characteristics

Derived from parental wild-type AA8 cells

Modest ionizing radiation sensitivity

Extreme crosslinking sensitivity (mitomycin C)

Chromosomally unstable

Deficient in Rad51 foci formation in response to damage

Complementable by human XRCC3 protein

GREEN FLUO SCENT PROTEIN

I-SC

E I

REEN FLUORESCENT PRO

Recombinational repair reporterRecombinational repair reporter

I-SceIendonuclease

GENE CONVERSION PRODUCT

GREEN FLUORESCENT PROTEIN REEN FLUORESCENT PRO

OR

END-JOINING PRODUCT

GREEN FLUQJSCENT PROTEIN REEN FLUORESCENT PRO

XRCC3 is a component of homologous recombination

WildType

NoI-SceI I-SceI

0.01% 5.64%

irs1SF

0.00% 0.22%

I-SceI+ XRCC3

4.92%

5.46%

Andrew J. Pierce, Roger D. Johnson and Maria Jasin.XRCC3 promotes homology-directed repair of DNA damage in mammalian cells.

Genes and Development 13(20): 2633-2638.

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Molecular Cell, Vol 7, 263-272, February 2001BRCA2 Is Required for Homology-Directed Repair of Chromosomal Breaks

Mary Ellen Moynahan, Andrew J. Pierce, and Maria Jasin

Truncated BRCA2(missing NLS)

WT

A cancer connection to recombination?

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Rad51 interacts with BRCA2

Nature 420, 287 - 293 (21 November 2002)Insights into DNA recombination from the structure of a RAD51–BRCA2 complex

LUCA PELLEGRINI, DAVID S. YU, THOMAS LO, SHUBHA ANAND, MIYOUNG LEE, TOM L. BLUNDELL & ASHOK R. VENKITARAMAN

Stereo diagram of Rad51 (red) and RecA (green) ATPase domains

Rad51 bound to BRC repeat peptide

BRCA2 has single-strand and double-strand DNA binding domains

Science, Vol 297, Issue 5588, 1837-1848, 13 September 2002 BRCA2 Function in DNA Binding and Recombination from a BRCA2-DSS1-ssDNA Structure

Haijuan Yang, Philip D. Jeffrey, Julie Miller, Elspeth Kinnucan, Yutong Sun,Nicolas H. Thomä, Ning Zheng, Phang-Lang Chen, Wen-Hwa Lee, Nikola P. Pavletich

Bind dsDNA

ssDNA

A. Deviren · N. Yalman · S. HacihanefiogluDifferential diagnosis of Fanconi anemia by nitrogen

mustard and diepoxybutaneAnn Hematol (2003) 82:223–227

Fanconi Anemia cell treated with nitrogen mustard

Fanconi Anemia cell treated with diepoxybutane

Fanconi Anemia

• pancytopenia• pigmentary changes in the skin• malformations of the heart, kidney, and limbs• leukemia

• FancD1 is BRCA2 (?)• FancD2 interacts with BRCA1

Amor-Gueret M. Bloom's syndrome. Orphanet Encyclopedia. February 2004

Normal cell metaphase Bloom's syndrome cell metaphase

Bloom's Syndrome

• proportionate pre- and postnatal growth deficiency• sun-sensitive• telangiectatic• hypo- and hyperpigmented skin• chromosomal instability, predisposition to malignancy

BLM protein in RecQ-helicase family with Werner Syndrome (WRN), Rothmund-Thomson Syndrome (RecQ4), and two other non-disease-associated members RecQL, RecQ5

Nature 418, 562 - 566 (01 August 2002)The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and

repairKARL-PETER HOPFNER, LISA CRAIG, GABRIEL MONCALIAN, ROBERT A. ZINKEL, TAKEHIKO USUI,

BARBARA A. L. OWEN, ANNETTE KARCHER, BRENDAN HENDERSON, JEAN-LUC BODMER, CYNTHIA T. MCMURRAY, JAMES P. CARNEY, JOHN H. J. PETRINI & JOHN A. TAINER

Rad50/Mre11/Nbs1

Rad50 (yellow/orange)Mre11 (blue circles)Zn+2 (red spheres)

Mre11 deficiency• ATLD (ataxia-telangiectasia-like disorder)• Radiosensitivity, spontaneous chromosome breakage, radioresistant DNA synthesis

Nbs1 deficiency• Nijmegen Breakage Syndrome (was Berlin Breakage Syndrome)• similar phenotype to ATLD

Nbs1 in here somewhere

Rad50 deficiency• no human patients identified

Electron micrographs

Crystal structure