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The Role of RecA in DNA Replication. Alastair Plant Salah Awad. 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).

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The role of reca in dna replication

The Role of RecA in DNA Replication

Alastair Plant

Salah Awad

MCB 720 - Winter 2011

January 20th, 2011


Discovery of reca
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 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): 1161–1168

Images from Cox (2007). Nat. Rev. Mol. Cell. Bio.


Reca dna binding
RecA-DNA binding

  • RecA preferentially binds to ssDNA.

  • It polymerises into a nucleoprotein filament.

  • Each monomer spans several nucleotide bases.

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 function
RecA function

RecA 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 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


Strand Exchange

RecA promotes strand exchange, allowing DNA polymerases to use homologous DNA as a template for repairing breaks and lesions.

RecA forms specific complexes with other Rec proteins depending upon the cause of replication fork stall.

Cox, Goodman, Kreuzer, Sherratt, Sandler and Marlans (2000). The Importance of Repairing Stalled Replication Forks. Nature 404 pp37-41


Dna binding pathway for the reca protein
DNA binding pathway for the RecA protein

  • DNA binding includes distinct nucleation

  • Filament Extension

  • Filament Dissociation

    proceeds 5’ to 3’

Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100


The repair of stalled replication forks
The Repair of Stalled Replication Forks

  • A- Strand

    Break

  • B- Blocking

    Lesion

Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100


The reca redistribution model
The RecA Redistribution Model

  • Duplex DNA is paired with the RecA-ssDNA

  • Strand exchange proceeds up

  • Dissociation of RecA protein

  • Release underwound DNA

Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100


Facilitated dna rotation model for reca protein mediated dna strand exchange
Facilitated DNA rotation model for RecA protein–mediated DNA strand exchange

  • Absence and

    Presence of

    ATP hydrolysis

Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100


An indirect helicase function of reca protein
An indirect helicase function of RecA protein DNA strand exchange

Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100


3 end invasion
3’ End Invasion DNA strand exchange

  • RecA protein promotes 3 end invasion.

  • Free dsDNA end processed by RecBCD enzyme

  • RecBCD generates a 3 single-strand extension.

  • RecBCD enzyme loads RecA protein onto the single-stranded DNA

  • 3’ End can be used as a replication primer.

    5’ End Invasion

  • 5’ End invasion could come in other form.

  • Assembly/disassembly process creates a difference between 3’ and 5’ ends.


Summary
Summary DNA strand exchange

  • RecA 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.


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