alastair plant salah awad mcb 720 - winter 2011 january 20 th, 2011
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The Role of RecA in DNA Replication
Alastair PlantSalah Awad
MCB 720 - Winter 2011January 20th, 2011
Discovery of RecA
Clark (1967) screened E. coli colonies for mutants with impaired recombination.
These mutants were UV-sensitive.
Homologous genes exist in prokaryotes and eukaryotes (e.g.Rad51).
AJ Clark (1967). The Beginning of a Genetic Analysis of Recombination Proficiency. J. Cell. Physiol.7, 0: Sup. 2 165-180.
RecA protein structure
RecA has two DNA-binding sites1:The primary site binds ssDNA.The secondary site binds dsDNA, allowing heteroduplex formation.
RecA uniquely performs ATP hydrolysis.
1A V Mazin and S C Kowalczykowski (1998). EMBO J. February 16; 17(4): 1161–1168Images from Cox (2007). Nat. Rev. Mol. Cell. Bio.
RecA-DNA binding
Qun Shan, Julie Bork, Inman and Cox. J. Mol. Biol. 1997 265 519-54, referenced at http://www.biochem.wisc.edu/faculty/inman/empics/dna-prot.htm 16th January 2011
RecA preferentially binds to ssDNA. It polymerises into a nucleoprotein filament. Each monomer spans several nucleotide bases.
RecA functionRecA rescues stalled replication forks by several methods:
Induction of the SOS reponse by assisting LexA autocatalysis1
Promotion of mutagenic polV-mediated TLS2
Replication fork regression2
DNA synapsis1
1Alberts et al. (2008). Garland Science. 2Lusetti and Cox (2002). The Bacterial RecA Protein and the Recombinational DNA Repair of Stalled Replication Forks. Annu. Rev. Biochem. 2002. 71:71–100
SOS and TLS SOS:Exposure to ultraviolet light induces the SOS response.RecA is required for UV-induced cleavage of the LexA repressor1.Repression of 43 SOS response genes is lifted2.
TLS:Trans-lesion synthesis is performed by Pol V, a low fidelity DNA polymerase.RecA lifts repression of UmuDC genes and cleaves the UmuD protein to form UmuD’, AKA Pol V2.
1Alberts et al. (2008). Garland Science. 2 Patel, Jiang, Woodgate, Cox and Goodman (2010). Critical Reviews in Biochemistry and Molecular Biology 45(3):171-184
Cox, Goodman, Kreuzer, Sherratt, Sandler and Marlans (2000). The Importance of Repairing Stalled Replication Forks. Nature 404 pp37-41
Strand Exchange
RecA forms specific complexes with other Rec proteins depending upon the cause of replication fork stall.
RecA promotes strand exchange, allowing DNA polymerases to use homologous DNA as a template for repairing breaks and lesions.
DNA binding pathway for the RecA protein
DNA binding includes distinct nucleationFilament ExtensionFilament Dissociation proceeds 5’ to 3’
Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100
The Repair of Stalled Replication Forks
A- Strand BreakB- Blocking Lesion
Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100
The RecA Redistribution ModelDuplex DNA is paired with the RecA-
ssDNAStrand exchange proceeds upDissociation of RecA proteinRelease underwound DNA
Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100
Facilitated DNA rotation model for RecA protein–mediated DNA strand exchange
Absence and Presence ofATP hydrolysis
Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100
An indirect helicase function of RecA protein
Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100
3’ End InvasionRecA protein promotes 3 end invasion.Free dsDNA end processed by RecBCD
enzymeRecBCD generates a 3 single-strand
extension.RecBCD enzyme loads RecA protein onto the
single-stranded DNA3’ End can be used as a replication primer.
5’ End Invasion5’ End invasion could come in other form. Assembly/disassembly process creates a
difference between 3’ and 5’ ends.
SummaryRecA is a multifunctional DNA-binding
protein.It polymerizes into a nucleoprotein filament.It induces the SOS response and promotes
mutagenic Trans-Lesion Synthesis by Pol V.It has ATP hydrolysis activity, enabling fork
migration.Four-strand exchange requires a helicase-like
activity of RecA.