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.
Highlights
Human Rad51 (RAD51), analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA in the presence of certain nucleotide cofactors
We show that unwinding of duplex DNA by RAD51 correlates positively with aggregation of RAD51-double stranded DNA (dsDNA) complexes
Further quantification of the gel revealed, maximal DNA unwinding (~44%) even at the lowest RAD51 concentration [3 μM] in the presence of Adenosine triphosphate (ATP)/Adenosine monophosphate (AMP)-PNP, whereas DNA unwinding increased as a function of RAD51 concentration in Adenosine 5'-O-(3-thiotriphosphate) (ATPγS)/Adenosine diphosphate (ADP) conditions and reached about 34% and 22% respectively at maximal protein concentration (9 μM) (Fig. 1I)
Summary
Human Rad (RAD51), analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA (dsDNA) in the presence of certain nucleotide cofactors. RAD51 recombinase, the eukaryotic homolog of RecA, is the core component in homologous recombination (HR) It performs fundamental roles such as homologous pairing and strand exchange in repairing DNA double strand breaks [1,2]. Rad binds both ssDNA/dsDNA [3,4,5] and forms right-handed helical nucleoprotein filaments in which DNA is extended 1.5 times that of B form DNA [4,6,7,8]. Based on E. coli RecA system, it is surmised that unwinding associated with dsDNA-protein filament is important in four-stranded pairing and strand exchange processes [11]. The first and (page number not for citation purposes)
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