Heteroduplex joint formation free of net topological change by Mhr1, a mitochondrial recombinase

Abstract

Homologous pairing, an essential process for homologous recombination, is the formation of a heteroduplex joint by an invading single-stranded DNA tail and a complementary sequence within double-stranded DNA (dsDNA). The base rotation of the parental dsDNA, to switch from parental base pairs to heteroduplex ones with the invading single-stranded DNA, sterically requires vertical extension between adjacent base pairs, which inevitably induces untwisting of the dsDNA. RecA is a prototype of the RecA/Rad51/Dmc1 family proteins, which promote ATP-dependent homologous pairing in homologous DNA recombination in vivo, except in mitochondria. As predicted by the requirement for the untwisting, dsDNA bound to RecA is extended and untwisted, and homologous pairing by RecA in vitro is extensively stimulated by the negative supercoils of dsDNA substrates. D-loop formation in negatively supercoiled dsDNA, which serves as an assay for homologous pairing, is also catalyzed in an ATP-independent manner by proteins structurally unrelated to RecA, such as Mhr1. Mhr1 is required for yeast mitochondrial DNA recombination instead of RecA family proteins. Inconsistent with the topological requirements, tests for the effects of negative supercoils revealed that Mhr1 catalyzes homologous pairing with relaxed closed circular dsDNA much more efficiently than with negatively supercoiled dsDNA. Topological analyses indicated that neither the process nor the products of homologous pairing by Mhr1 involve a net topological change of closed circular dsDNA. This would be favorable for homologous recombination in mitochondria, where dsDNA is unlikely to be under topological stress toward unwinding.Wepropose a novel topological mechanism wherein Mhr1 induces untwisting without net topological change. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.