Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination.

ABSTRACT: Recombinases polymerize along single-stranded DNA (ssDNA) at the end of a broken DNA to form a helical nucleofilament with a periodicity of ?18 bases. The filament catalyzes the search and checking for homologous sequences and promotes strand exchange with a donor duplex during homologous recombination (HR), the mechanism of which has remained mysterious since its discovery. Here, by inserting mismatched segments into donor duplexes and using single-molecule techniques to catch transient intermediates in HR, we found that, even though 3 base pairs (bp) is still the basic unit, both the homology checking and the strand exchange may proceed in multiple steps at a time, resulting in ?9-bp large steps on average. More interestingly, the strand exchange is blocked remotely by the mismatched segment, terminating at positions ?9 bp before the match-mismatch joint. The homology checking and the strand exchange are thus separated in space, with the strand exchange lagging behind. Our data suggest that the strand exchange progresses like a traveling wave in which the donor DNA is incorporated successively into the ssDNA-RecA filament to check homology in ?9-bp steps in the frontier, followed by a hypothetical transitional segment and then the post-strand-exchanged duplex.

SUBMITTER: Huang X 

PROVIDER: S-EPMC7456136 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature