Transcript RAD51

MCB 720
Mechanisms of Homologous
Recombination & Protein
Rad51
Jia Liu
Adam Jara
Jan 20th, 2011
Outline
• I. Introduction
• II. Mechanisms of Homologous
Recombination (HR)
• III. HR in Saccharomyces cerevisiae
(SC);
• IV. Rad 51 protein;
• V. Conclusion.
I. Introduction
• HR allows for proper chromosome
segregation during meiosis, promoting
genomic integrity between generations.
• Represents the highest fidelity repair
mechanism for DNA breaks.
• Diploid human cell maintains ~6x109 bp of
DNA.
• Highly regulated process, as too much HR
can lead to large chromosomal
rearrangements.
Double Strand Break Repair
• Unintended DSB represents on of the
most toxic DNA lesions and can result in
chromosomal rearrangement.
• These can occur from environmental
stresses: ie. ionizing radiation,
pharmaceutics.
• Mammalian cells have three main
mechanisms of DSB repair: nonhomologous end joining, single strand
annealing, and HR.
Pathways of DSB Repair
•The primary pathway in
eukaryotes for repairing
DSB is NHEJ
•NHEJ can lead to gross
genetic rearrangment
and loss of genomic
material
•SSA depends on
tandem repeats and can
also lead to loss of
genomic material
•Only HR offers the
capacity to resynthesize
damaged or missing
sequence. A template is
necessary
Heyer W, Ehmsen KT, Jie L. (2010) Annu. Rev. Genetics 44: 113-39
Regulation of Homologous Recombination
•During S phase sister
chromatids are present
and are the best
substrate for HR
•CDKs present during
S phase phosphorylate
CtIP.
•CtIP mediates 5’ end
resection allowing for 3’
overhangs
•Unphosphorylated
BRCA2 no longer
inhibits Rad51.
Heyer W, Ehmsen KT, Jie L. (2010) Annu. Rev. Genetics 44: 113-39
•Rad51 forms
nucleofilaments with
the 3’ overhangs and
promotes HR.
Synthesis Dependent Strand Annealing (SDSA)
•Principal mechanism for DSB repair by
HR. Reduces the potential for risk of
crossover.
•The MRN (MRE11, NBS1, RAD50)
complex recognizes the DSB and
processes the 3’ overhang which is initially
bound by RPA.
•RAD52 and BRCA2 mediate RPA
dissociation and RAD51 nucleofilament
formation.
•RAD51 performs strand invasion and
homology search forming a displacement
(D-) loop.
•DNA is synthesized by DNA Pol η
(mammals)
•New strand dissociates and binds to
original 3’ end. Gap is filled by DNA ligase.
Lodish et al., Molecular Cell Biology, 5th ed. Fig 23-31
Double Holliday Junction (dHJ)
•Most likely mechanism by which crossover
occurs.
•Involves the strand invasion by both 3’
RAD51 nucleofilaments.
•Resolution of the heteroduplexes (Holliday
Junctions) by many different
endonucleases (MUS81-EME1 , GEN1,
TOP1).
•Directionality of resolution determines
gene conversion or crossover.
Helleday T, Lo J, van Gent D, Engelward B. (2007) DNA Repair 6: 923-35.
Loss of HR Regulation
• BRCA2 facilitates displacement of RPA
and loading of Rad51 onto ssDNA.
– Loss of function leads to chromosome
abnormalities.
• BLM functions as a 3’->5’ helicase in
presynaptic events
– Mutants lead to Bloom’s Syndrome, a
condition characterized by a predisposition to
cancer and loss of heterozygosity.
Major Proteins Involved in HR
•HR requires 3’
overhang that
constitutes the
presynaptic filament
•DNA Pol is
resposible for
forming the
completary strands
•Holliday
heteroduplexes
must be resolved.
Hinz J. (2010) Environmental and Molecular Mutagenesis 51:582-603.
III. HR in Saccharomyces
cerevisiae (SC)
• Saccharomyces cerevisiae;
• RAD 52 epistasis group: RAD50, RAD51,
RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/TID1,
MRE11 and XRS2 genes;
• RAD 51 family;
• RAD51: wild type gene or locus; rad51:
the mutant gene or locus; Rad51: protein.
http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae, accessed on Jan 17th, 2011.
Alan Wheals. Scanning Electron Micrograph of Saccharomyces cerevisiae.
IV. Rad 51 in HR of S.
cerevisiae
A. Mutant phenotype
• S. cerevisiae rad51 mutants;
• Sensitive to IR; reduce mitotic and meiotic
recombination;
• Chemical agents; anticanter drugs;
• Affect recombination events;
• Meiosis.
B. Gene and protein
• ScRad51;
• 400 A.A., 43kDa;
• 3 regions: N terminal, C terminal, central
core;
• A putative leucine zipper motif;
• Rad51-dependent pathways: DSB-induced
gene conversion;
• Rad51-independent pathways:
spontaneous recombination, DSB-induced
DNA replication (BIR).
ScRad51: Homologue of Rec A
•
•
•
•
Structures similarities;
Main RecA-like recombinase;
ATP-dependent DNA-binding activity;
Difference:
Requirements for initiating joint molecules
and the polarity of branch migration.
• Rec A: ssDNA;
• ScRad 51:dsDNA with ssDNA tails.
C. Interacting partners
• Interactions with multiple proteins;
• ScRad 51: ScRad 51, Rad52, Rad 54,
Rad55, Rdh54/Tid1(Rad54), Sgs1.
• Complex involving a large number of
proteins “recombinosome” .
C. Interacting partners
Homologous recombination factors in Saccharomyces cerevisiae
S.
Cerevisiae
Biochemical activity/function
Rad51
ATP-dependent DNA-binding
activity;
ATP-dependent homologous
pairing and DNA strand
exchange activities
Interacting partners
Yeast: Rad52, Rad54, Rad55,
Rdh54/Tid1, Sgs1, Rsi1/Apc2, Zip3,
Dmc1*, Mlh1, Sap1, Ubc9,
YMR233W,
YPL238C, YPR011C; human:
Rad51C,
Rad52, Rad54, Rad54B*, Werner’s
syndrome protein, Xrcc3, c-Abl, p53,
Brca1*, Brca2, Ube2I/Ubc9, Ubl1,
Pir51, Rpb1.
Dudas A, Chovanec M. (2004) Mutation Research 566: 131-167.
Protein–protein interaction network
(A) mitosis
(B) meiosis
Dudas A, Chovanec M. (2004) Mutation Research 566: 131-167.
V. Conclusion
•HR is the pathway by which genome integrity is
maintained
•High fidelity pathway for DSB repair
•Drive genetic recombination
•The main HR pathway involved in DSB repair is SDSA
•The dHJ pathway likely allows for mitotic crossover
events
•The protein involved in eukaryotes HR is Rad51 (RecA
homolog), which forms complex with multiple proteins,
promoting homologous pairing and DNA strand
exchange activities in HR.
References
•Dudas A, Chovanec M. (2004) DNA double-strand break repair by
homologous recombination. Mutation Research 566: 131-167.
•Helleday T, Lo J, van Gent DC, Engelward BP. (2007) DNA double-strand
break repair: From mechanistic understanding to cancer treatment. DNA
Repair 6: 923-935
•Heyer W, Ehmsen KT, Liu J. (2010) Regulation of Homologous
Recombination in Eukaryotes. Annu. Rev. Genet. 44: 113-39.
•Hinz JM. (2010) Role of Homologous Recombination in DNA Interstrand
Crosslink Repair. Environmental and Molecular Mutagenesis 51: 582-603.