Role of MutS ATPase Activity in MutS,L-dependent Block of in Vitro Strand Transfer

Abstract

In addition to mismatch recognition, Escherichia coliMutS has an associated ATPase activity that is fundamental to repair. Hence, we have characterized two MutS mutant gene products to define the role of ATP hydrolysis in homeologous recombination. These mutants, denoted In addition to its role in replication fidelity, mismatch repair contributes to genome stability by controlling the level of recombination between closely related sequences (1-9). In Escherichia coli, MutS and MutL act to block recombinant yield when phenotypic selection arises in the vicinity of heteroallelic markers (10). Recent work from Zahrt and Maloy (11) provided more evidence implicating mismatch repair in recombination. Specifically, they showed that the efficiency of chromosomal gene transfer between Salmonella typhimurium and the closely related Salmonella typhi is low due to mismatch repair. Zahrt and Maloy (11) attributed this barrier of genetic exchange to DNA divergence and the ability of MMR to correct this event in favor of the recipient strand. Abdulkarim and Hughes (12) also showed that the mutHLSU genes increased the fidelity of exchange 1000-fold between the TufA and TufB translation factors, whose sequences share 99% sequence identity. Mismatch repair in this case ensures that the integrity of the genome is preserved by allowing these genes to evolve in concert.The first biochemical evidence to implicate mismatch repair in homologous recombination was provided by the in vitro RecA-catalyzed three-strand transfer reaction. E. coli MutS was shown to inhibit exchange between M13 and fd DNAs whose sequence heterology is ϳ3% at the nucleotide level (13). Since MutS was without effect on M13-M13 or fd-fd exchange, we attribute the block to the occurrence of mismatched base pairs in newly formed heteroduplex.MutL enhanced the MutS-dependent block of M13-fd exchange. Indeed, these proteins completely abolished full-length heteroduplex between these DNAs and is reminiscent of the proposed role of these activities in . MutL is believed to add to the MutS mismatch complex in an ATP-dependent fashion. Evidence for this stems from work by Grilley et al. (20), who showed the region of DNase I protection by MutS and ATP is extended by MutL. In the experiments described here, the role of ATP hydrolysis by MutS is examined on RecA-strand transfer. Previous work by Wu and Marinus (21) described dominant negative mutants of MutS from MNNG treatment and showed a large fraction of the isolates were missense mutations within the nucleotide binding domain (21). It is here that we have characterized two of these mutants, MutS501 and MutS506, and show that 1) mismatch binding is retained, 2) ATP hydrolysis is reduced, and 3) mismatch correction with MutS Ϫ extracts is not restored. These mutants also blocked RecA-catalyzed strand transfer between M13 and fd DNAs. That MutS501, and to a lesser extent MutS506, inhibited M13-fd and not M13-M13 exchange, suggests that mismatch recognition precludes RecA-dependent branch migration in regions of nonhomo...

The Escherichia coliMutL Protein Physically Interacts with MutH and Stimulates the MutH-associated Endonuclease Activity

“…This ATP binding requirement likely is not caused solely by MutS for the following reason. The K m for ATP hydrolysis catalyzed by our purified MutS preparation was 23 M (data not shown), consistent with a recent report (26). The K m for ATP during the overall mismatch repair reaction in E. coli is approximately 300 M (4).…”

Impact of mutS Inactivation on Foreign DNA Acquisition by Natural Transformation in Pseudomonas stutzeri

“…Such extensions are catalyzed by RecA and facilitated by RuvAB to cover stretches that are 1 kbp or more long (1,33). MutS was shown to impede strand exchange and branch migration between divergent DNA sequences by RecA (87,88). Similarly, branch migration through regions of heterology stimulated by RuvAB was prevented through the MutSL complex (21).…”

“…Thus, the sexual isolation of strains defective in mismatch repair is decreased (13,46). The antirecombination effect of the MMR system seen in vivo was explained by the abortion of strand exchange reactions with MutS and MutL demonstrated in vitro (87,88). Therefore, the MMR system controls not only the level of spontaneous mutability of cells but also the level of recombinative acquisition of new genetic information by homologous recombination (9,81).…”

Genetic Exchange Between Homeologous Sequences in Mammalian Chromosomes Is Averted by Local Homology Requirements for Initiation and Resolution of Recombination

“…Our current work does not directly address the role of MMR in hDNA rejection, but our experiments with pHYB121 led us to conclude that rapid action of MMR on mismatches in hDNA is not a suitable explanation for dismantling hDNA. It has been shown that MMR in bacteria reduces the rate of strand exchange in the presence of mismatches (Worth et al 1994(Worth et al , 1998Bazemore et al 1997;Zahrt and Maloy 1997;Fabisiewicz and Worth 2001) but, importantly, MMR does not target preformed mismatched hDNA (Worth et al 1994;Westmoreland et al 1997). Work done in yeast also provides evidence that MMR proteins play a role in impeding branch migration through mismatches Jinks-Robertson 1998, 1999).…”