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.