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Recombinational Repair
Andrew Pierce
Graduate Center for Toxicology
University of Kentucky
TOX 680
Recombination: bringing DNA strands together in new ways
Strand annealing
Spontaneous or
Rad52 mediated
Strand invasion
RecA or
Rad51 mediated
Recombination is used to repair topological damage
Single-strand gap repair
Double-strand break repair
Recombination is used to repair topological damage
Ends need to
invade the duplex
Replication across a nicked template
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, 1998
Taro Nishinaka, Akira Shinohara, Yutaka Ito, Shigeyuki Yokoyama, and Takehiko Shibata
Rad51 is required for cellular viability
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
4 Gy g
Rad51-
The EMBO Journal (1998) 17, 598–608 Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death
Eiichiro 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
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
A:
B:
C-D:
E:
Untreated
4 Gy g (+3h)
4 Gy g (+8h)
Rad51- (+21h)
The EMBO Journal (1998) 17, 598–608 Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death
Eiichiro Sonoda, Masao S. Sasaki, Jean-Marie Buerstedde, Olga Bezzubova, Akira Shinohara,
Hideyuki Ogawa, Minoru Takata, Yuko Yamaguchi-Iwai and Shunichi Takeda
Rad51 paralog family
Rad51: ATP-hydrolysis-mediated strand exchange
DMC1: meiotic paralog
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Nature Reviews Cancer 2; 351-360 (2002)
ONCOGENIC TYROSINE KINASES AND THE DNA-DAMAGE RESPONSE
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
I-SCE I
Recombinational repair reporter
SCENT PROTEIN
REEN FLUORESCENT PRO
I-SceI
endonuclease
GREEN FLUQJSCENT PROTEIN
REEN FLUORESCENT PRO
END-JOINING PRODUCT
OR
GREEN FLUORESCENT PROTEIN
REEN FLUORESCENT PRO
GENE CONVERSION PRODUCT
XRCC3 is a component of homologous recombination
No
I-SceI
I-SceI
+ XRCC3
I-SceI
Wild
Type
0.01%
5.64%
4.92%
0.00%
0.22%
5.46%
irs1SF
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.
A cancer connection to recombination?
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
WT
Truncated BRCA2
(missing NLS)
Molecular Cell, Vol 7, 263-272, February 2001
BRCA2 Is Required for Homology-Directed Repair of Chromosomal Breaks
Mary Ellen Moynahan, Andrew J. Pierce, and Maria Jasin
Rad51 interacts with BRCA2
Rad51 bound to
BRC repeat peptide
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Stereo diagram of
Rad51 (red) and RecA
(green) ATPase domains
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
BRCA2 has single-strand and double-strand DNA binding domains
Bind dsDNA
ssDNA
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
Fanconi Anemia cell treated with nitrogen mustard
Fanconi Anemia
• pancytopenia
• pigmentary changes in the skin
• malformations of the heart, kidney, and limbs
• leukemia
• FancD1 is BRCA2 (?)
• FancD2 interacts with BRCA1
Fanconi Anemia cell treated with diepoxybutane
A. Deviren · N. Yalman · S. Hacihanefioglu
Differential diagnosis of Fanconi anemia by nitrogen mustard and
diepoxybutane
Ann Hematol (2003) 82:223–227
Bloom's Syndrome
Normal cell metaphase
Bloom's syndrome cell metaphase
Amor-Gueret M. Bloom's syndrome. Orphanet Encyclopedia. February 2004
• 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, RecQ5b
Rad50/Mre11/Nbs1
Crystal structure
Rad50 deficiency
• no human patients identified
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 (yellow/orange)
Mre11 (blue circles)
Zn+2 (red spheres)
Electron micrographs
Nature 418, 562 - 566 (01 August 2002)
The Rad50 zinc-hook is a structure joining Mre11 complexes in DNA recombination and repair
KARL-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