Interaction of Escherichia coli MutS and MutL at a DNA Mismatch

Abstract

MutS and MutL are both required to activate downstream events in DNA mismatch repair. We examined the rate of dissociation of MutS from a mismatch using linear heteroduplex DNAs or heteroduplexes blocked at one or both ends by four-way DNA junctions in the presence and absence of MutL. In the presence of ATP, dissociation of MutS from linear heteroduplexes or heteroduplexes blocked at only one end occurs within 15 s. When both duplex ends are blocked, MutS remains associated with the DNA in complexes with half-lives of 30 min. DNase I footprinting of MutS complexes is consistent with migration of MutS throughout the DNA duplex region. When MutL is present, it associates with MutS and prevents ATP-dependent migration away from the mismatch in a manner that is dependent on the length of the heteroduplex. The rate and extent of mismatch-provoked cleavage at hemimethylated GATC sites by MutH in the presence of MutS, MutL, and ATP are the same whether the mismatch and GATC sites are in cis or in trans. These results suggest that a MutS-MutL complex in the vicinity of a mismatch is involved in activating MutH.Misincorporation of bases during DNA replication that escapes proofreading results in the formation of mismatches, either unpaired or mispaired bases. The DNA mismatch repair pathway targets these mismatches for correction and contributes as much as 1000-fold to the overall fidelity of DNA replication (reviewed in Refs. 1 and 2). The importance of this repair pathway is highlighted by the finding that mutations in mismatch repair genes segregate with the cancer predisposition syndromes hereditary nonpolyposis colon cancer and familial colorectal cancer (reviewed in Refs. 3 and 4). In addition, inactivation of mismatch repair genes by promoter methylation has been documented in some sporadic tumors (reviewed in Ref. 1).Much of our current understanding of mismatch repair stems from studies of the Escherichia coli methyl-directed mismatch repair pathway where repair has been reconstituted in vitro using purified proteins (1, 2). MutS recognizes and binds to seven of eight possible base pair mismatches as well as one to four unpaired bases. These mismatches arise by misincorporation of deoxynucleotides or template slippage, respectively. In addition to binding DNA, all MutS proteins have an intrinsic ATPase activity and are members of the ATP binding cassette transporter superfamily (5). Recognition of the mismatch by MutS triggers subsequent steps of mismatch repair in which MutS together with MutL protein and ATP activates an endonuclease, MutH, that cleaves a transiently unmethylated daughter strand at hemimethylated GATC sites, thereby conferring strand specificity on the repair process. The mismatchprovoked strand scission constitutes a point of entry for helicase II (UvrD) and exonucleases that excise the daughter strand in the region spanning the GATC site and the mis- A critical problem in mismatch repair is understanding how binding to a mismatch by MutS triggers downstream repair events such as the activat...

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…In addition, DNasel footprinting studies reveal that whereas MutS protects approximately one turn of the DNA helix to either side of the mismatch consistent with the crystal structures, the MutS-MutL interaction in the presence of ATP yields a very large DNasel footprint indicative of multiple proteins bound to the DNA (Grilley et al, 1989;Selmane et al, 2003). Formation of a ternary complex involving either the E. coli or human MMR proteins also requires DNA heteroduplexes in excess of 60 bp reflecting multiple binding events in vitro (Blackwell et al, 2001;Plotz et al, 2002;Schofield et al, 2001b). Whether this is an artefact of in vitro conditions or reflects multiple loadings of MMR proteins, either multiple loading of diffusing clamps or cooperative binding of a protein filament, is not definitively established.…”

Asymmetric ATP Binding and Hydrolysis Activity of the Thermus aquaticus MutS Dimer Is Key to Modulation of Its Interactions with Mismatched DNA

“…Several research groups have reported an apparent reduction in the affinity of MutS for mismatched DNA following ATP binding, which manifests as MutS sliding off short linear mismatched DNA substrates with unblocked ends during gel mobility-shift analysis (1,13,(23)(24)(25)28,48); presumably, such movement of MutS on DNA serves an important function during mismatch repair. Since we have now identified some nucleotide-bound forms of MutS in the pathway, we tested their interaction with +T-containing duplex DNA substrate.…”

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

“…ATP binding and hydrolysis appear to modulate the interactions between MutS/Msh and DNA as well as other proteins in the repair pathway; thus, understanding how MutS/Msh proteins utilize ATP is necessary for understanding how they function in DNA mismatch repair. Several model mechanisms have been proposed for MutS/Msh action upon mismatch recognition: (a) MutS/Msh proteins translocate on DNA, fuelled by ATP binding and hydrolysis, possibly to interact with other proteins on DNA and coordinate mismatch recognition with downstream events such as initiation of strand excision and DNA resynthesis [8][9][10]; (b) upon binding ATP MutS/Msh proteins form sliding clamps that diffuse freely on DNA, again, to contact downstream repair proteins and direct repair [11,12]; (c) MutS/Msh proteins utilize ATP binding and hydrolysis to modulate their interaction with DNA, while remaining at the mismatch to direct repair [13][14][15][16][17]. At present, experimental data are available in support of each of these very different model mechanisms, therefore the investigation into MutS/Msh DNA binding and ATPase activities continues.…”