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BMC Biochemistry

, 10:2

First Online: 09 January 2009Received: 28 May 2008Accepted: 09 January 2009

Abstract

BackgroundHuman Rad51 RAD51, analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA dsDNA in the presence of certain nucleotide cofactors. ATP hydrolysis is not required for this process, because even ATP non hydrolysable analogs like AMP-PNP and ATPγS, support DNA unwinding. Even ADP, the product of ATP hydrolysis, feebly supports DNA unwinding.

ResultsWe find that human Rad52 RAD52 stimulates RAD51 mediated DNA unwinding in the presence of all Adenine nucleotide cofactors, except in AMP and no nucleotide conditions that intrinsically fail to support unwinding reaction while enhancing aggregation of RAD51-dsDNA complexes in parallel. Interestingly, salt at low concentration can substitute the role of RAD52, in facilitating aggregation of RAD51-dsDNA complexes, that concomitantly also leads to better unwinding.

ConclusionRAD52 itself being a highly aggregated protein perhaps acts as scaffold to bring together RAD51 and DNA molecules into large co-aggregates of RAD52-RAD51-DNA complexes to promote RAD51 mediated DNA unwinding reaction, when appropriate nucleotide cofactors are available, presumably through macromolecular crowding effects. Our work highlights the functional link between aggregation of protein-DNA complexes and DNA unwinding in RAD51 system.

AbbreviationsRAD51human Rad51

RAD52human Rad52

HRHomologous Recombination

ssDNAsingle stranded DNA

dsDNAdouble stranded DNA

Topo ITopoisomerase I

ATPAdenosine triphosphate

ADPAdenosine diphosphate

AMPAdenosine monophosphate

ATPγSAdenosine 5-O-3-thiotriphosphate

AMP-PNPadenosine 5-β, γ-imido triphosphate

KClPotassium Chloride

BSABovine Serum Albumen

TLCThin Layer Chromatography

A600Absorbance at 600 nm

P MSFPhenyl Methyl Sulphonyl Fluoride.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2091-10-2 contains supplementary material, which is available to authorized users.

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Autor: Kamakshi Balakrishnan - Neeraja M Krishnan - Anagha Kulkarni - Basuthkar J Rao

Fuente: https://link.springer.com/article/10.1186/1471-2091-10-2



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