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The Role of RecA in DNA Replication

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1 The Role of RecA in DNA Replication
Alastair Plant Salah Awad MCB Winter 2011 January 20th, 2011

2 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 UV-sensitivity measured in terms of recovery after exposure – resumption of cell division. Lambda prophage induction failed in some recombination mutants; also X-ray sensitivity.

3 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. RecA identified from susceptibility to UV 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.

4 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 , referenced at 16th January 2011

5 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 :71–100 Replication fork regression assists gap repair

6 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): Rep fork regression assists gap repair

7 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

8 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

9 The Repair of Stalled Replication Forks
A- Strand Break B- Blocking Lesion Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100

10 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

11 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

12 An indirect helicase function of RecA protein
Lusetti and Cox, Annu. Rev. Biochem. (2002) ,71:71–100

13 3’ End Invasion 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.

14 Summary It polymerizes into a nucleoprotein filament.
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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