Abstract
Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.
Highlights
Unrepaired DNA double-strand breaks are associated with increased risk of certain cancers in humans [1,2]
The presynaptic filament is aligned with homologous regions of double-stranded DNA, and a strand exchange occurs in which the singlestranded DNA invades and disrupts the double-stranded DNA to exchange out one of the strands
Results of this study suggest that UvsX recombinase avoids inhibition by excess non-homologous double-stranded DNA (dsDNA) through allosteric effects mediated by single-stranded DNA (ssDNA) binding
Summary
Unrepaired DNA double-strand breaks are associated with increased risk of certain cancers in humans [1,2]. Homologous recombination (HR) is used by cells for error-free repair of DNA double-strand breaks, and to restart stalled replication forks [3,4]. Recombinases of the RecA/Rad family perform the central catalytic role in homologous recombination of DNA. Recombinases bind cooperatively to regions of single-stranded DNA (ssDNA) to form a presynaptic filament. The presynaptic filament is aligned with homologous regions of double-stranded DNA (dsDNA), and a strand exchange occurs in which the singlestranded DNA invades and disrupts the double-stranded DNA to exchange out one of the strands. The recombinase filament must dissociate from the DNA to allow for completion of the repair process [5,6]
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