Biochemical Characterization of the Human RAD51 Protein: III. MODULATION OF DNA BINDING BY ADENOSINE NUCLEOTIDES

Gregory Tombline,Christopher D Heinen,Kang-Sup Shim,Richard Fishel

doi:10.1074/jbc.m109917200

Gregory Tombline, Christopher D Heinen + Show 2 more

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https://doi.org/10.1074/jbc.m109917200

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Abstract

Adenosine nucleotides affect the ability of RecA small middle dotsingle-stranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. Here we have determined that ADP and ATP/ATPgammaS affect the DNA binding and aggregation properties of the human RecA homolog human RAD51 protein (hRAD51). These studies have revealed significant differences between hRAD51 and RecA. In the presence of ATPgammaS, RecA forms a stable complex with ssDNA, while the hRAD51 ssDNA complex is destabilized. Conversely, in the presence of ADP and ATP, the RecA ssDNA complex is unstable, while the hRAD51 ssDNA complex is stabilized. We identified two hRAD51 small middle dotssDNA binding forms by gel shift analysis, which were distinct from a well defined RecA small middle dotssDNA binding form. The available evidence suggests that a low molecular weight hRAD51 small middle dotssDNA binding form (hRAD51 small middle dotssDNA(low)) correlates with active ADP and ATP processing. A high molecular weight hRAD51 small middle dotssDNA aggregate (hRAD51 small middle dotssDNA(high)) appears to correlate with a form that fails to process ADP and ATP. Our data are consistent with the notion that hRAD51 is unable to appropriately coordinate ssDNA binding with adenosine nucleotide processing. These observations suggest that other factors may assist hRAD51 in order to mirror RecA recombinational function.

Highlights

Adenosine nucleotides affect the ability of RecA1⁄7singlestranded DNA nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination
We have developed a system in which a model oligonucleotide (oligo(dT)50) is biotinylated at either the 3Ј- or 5Ј-end and attached via a streptavidin linkage to an IAsys biosensor (IAB) cuvette coated with biotin
We obtained KD values for hRAD511⁄7oligo(dT)50 binding in the range of 78 –176 nM depending upon the magnesium concentration or whether the 5Ј- or 3Ј-biotinylated substrate was human RAD51 protein (hRAD51) DNA Binding examined (Table I)

Summary

Introduction

Adenosine nucleotides affect the ability of RecA1⁄7singlestranded DNA (ssDNA) nucleoprotein filaments to cooperatively assume and maintain an extended structure that facilitates DNA pairing during recombination. HRAD51 appears to lack ATP-induced cooperativity during ATP hydrolysis [11] and/or the ability to form extended/active hRAD511⁄7 ssDNA nucleoprotein filaments in the presence of ATP␥S [12]. These data suggest that hRAD51 differs significantly from RecA in its ability to couple ADP and ATP/ATP␥S processing with ssDNA interactions. Predicting the coordinate self-association, nucleotide binding/hydrolysis, and ssDNA binding activity of hRAD51 based on a comparison with the RecA protein structure is not straightforward. The RecA crystal structure indicated that residues involved in self-association, adenosine nucleotide binding/hydrolysis, and DNA binding appeared juxtaposed and coordinated [21, 22]. These residues appear critical for biological activity and probably function as allosteric effec-

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