The hRad51 and RecA proteins show significant differences in cooperative binding to single-stranded DNA

被引:55
作者
De Zutter, SK [1 ]
Knight, KL [1 ]
机构
[1] Univ Massachusetts, Med Ctr, Dept Biochem & Mol Biol, Worcester, MA 01655 USA
关键词
hRad51; protein; RecA protein; cooperative DNA binding; IAsys biosensor; homologous recombination;
D O I
10.1006/jmbi.1999.3200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The human Rad51 protein (hRad51), like its bacterial homologue RecA, catalyzes genetic recombination between homologous single and double-stranded DNA substrates. Using IAsys biosensor technology, we have examined the critical first step in this process, the binding of hRad51 and RecA to ssDNA. We show that hRad51 binds cooperatively and with high affinity to an oligonucleotide substrate in both the absence and presence of nucleotide cofactors. In fact, both ATP and ATP gamma S have a slight inhibitory effect on hRad51 binding affinity. We show that this results from a decrease in the intrinsic affinity of a given monomer for ssDNA, which is counterbalanced by an increase in the cooperative assembly of protein onto DNA. In contrast, we show that the dramatic Nm-induced increase in ssDNA binding affinity of RecA is accounted for by a significant increase in cooperative filament assembly and not by an increase in the intrinsic DNA binding affinity of monomeric RecA. These results demonstrate that although the hRad51 and RecA proteins display many structural and functional similarities, they show profound inherent mechanistic differences. (C) 1999 Academic Press.
引用
收藏
页码:769 / 780
页数:12
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