Abstract
The Escherichia coli RuvABC proteins process recombination intermediates during genetic recombination and recombinational repair. Although early biochemical studies indicated distinct RuvAB-mediated branch migration and RuvC-mediated Holliday junction resolution reactions, more recent studies have shown that the three proteins act together as a "resolvasome" complex. In this work we have used recombination intermediates made by RecA to determine whether the RuvAB proteins affect the sequence specificity of the RuvC resolvase. We find that RuvAB proteins do not alter significantly the site specificity of RuvC-dependent cleavage, although under certain conditions, they do affect the efficiency of cleavage at particular sites. The presence of RecA also influences cleavage at some sites. We also show that the RuvAB proteins act upon transient strand exchange intermediates made using substrates that have the opposite polarity of those preferred by RecA. Together, our results allow us to develop further a model for the recombinational repair of DNA lesions that lead to the formation of post-replication gaps during DNA replication. The novel features of this model are as follows: (i) the RuvABC resolvasome recognizes joints made by RecA; (ii) resolution by RuvABC occurs at specific sites containing the RuvC consensus cleavage sequence 5'-(A/T)TT downward arrow(G/C)-3'; and (iii) Holliday junction resolution often occurs close to the initiating gap without significant heteroduplex DNA formation.
Highlights
Several processes, including the repair of broken replication forks or DNA lesions, the reassortment of alleles, and the correct segregation of chromosomes during meiosis, employ the mechanism of homologous recombination
The novel features of this model are as follows: (i) the RuvABC resolvasome recognizes joints made by RecA; (ii) resolution by RuvABC occurs at specific sites containing the RuvC consensus cleavage sequence 5-(A/T)TT2(G/C)-3; and (iii) Holliday junction resolution often occurs close to the initiating gap without significant heteroduplex DNA formation
Since the ruvA and ruvB genes form an operon that is regulated by the SOS system and their expression is induced as a result of DNA damage [40, 41], it is possible that two types of complex might form in vivo as follows: (i) a RuvABC complex capable of promoting branch migration and resolution, and (ii) a subcomplex consisting of RuvAB alone, which would mediate only branch migration [31, 42]
Summary
Several processes, including the repair of broken replication forks or DNA lesions, the reassortment of alleles, and the correct segregation of chromosomes during meiosis, employ the mechanism of homologous recombination. Like RuvA, RuvC interacts with RuvB [31] and can target RuvB rings to the Holliday junction, resulting in a complex capable of both limited branch migration and cleavage [32]. Analysis of the RuvCG114D mutant protein has indicated that RuvC and RuvA interact physically on the Holliday junction, presumably by binding opposite faces [33]. These and other data derived from biochemical (34 –36), genetic [37, 38], and model building [11, 12, 39] studies support the proposal that RuvABC proteins form a Holliday junctionprocessing complex that mediates concerted branch migration and resolution.
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