Abstract
In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions.
Highlights
Homologous genetic recombination plays essential roles in DNA double-strand break repair to maintain genome stability and in genetic diversification, and defects in homologous recombination in mitotic cells cause carcinogenesis and various diseases
Our study revealed that RecA actively selects single-stranded DNA for the primary DNA binding and that Asp-161 in loop L1 plays a crucial role in this mechanism
The D161A mutation extensively enhanced double-stranded DNA binding with only a slight effect on single-stranded DNA binding shown by the electrophoresis mobility shift assay (Figure 2), and doublestranded DNA-dependent adenosine triphosphate (ATP) hydrolysis, by removing the effects of the presence or absence of supercoils (Figures 3 and 4A) and the stringent Mg2+ concentration requirements (Figure 3)
Summary
Homologous genetic (or DNA) recombination plays essential roles in DNA double-strand break repair to maintain genome stability and in genetic diversification, and defects in homologous recombination in mitotic cells cause carcinogenesis and various diseases (see [1,2,3,4] for reviews). Homologous joint formation is a crucial step in homologous recombination. In all organisms, this is catalyzed in an adenosine triphosphate (ATP)-dependent manner by the RecA-family recombinases, including bacterial RecA and eukaryotic Rad and Dmc, among which Escherichia coli (E. coli) RecA has been most thoroughly studied for decades ((5,6); see [7,8,9] for reviews).
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