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...

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The crystal structures of MutS protein from Thermus aquaticus and Escherichia coli in a complex with a mismatch-containing DNA duplex reveal that the Glu residue in a conserved Phe-X-Glu motif participates in a hydrogen-bonded contact with either an unpaired thymidine or the thymidine of a G-T base-base mismatch. Here, the role of hydrogen bonding in mismatch recognition by MutS is assessed. The relative affinities of MutS for DNA duplexes containing nonpolar shape mimics of A and T, 4-methylbenzimidazole (Z), and difluorotoluene (F), respectively, that lack hydrogen bonding donors and acceptors, are determined in gel mobility shift assays. The results provide support for an induced fit mode of mismatch binding in which duplexes destabilized by mismatches are preferred substrates for kinking by MutS. Hydrogen bonding between the O⑀2 group of Glu and the mismatched base contributes only marginally to mismatch recognition and is significantly less important than the aromatic ring stack with the conserved Phe residue. A MutS protein in which Ala is substituted for Glu 38 is shown to be defective for mismatch repair in vivo. DNA binding studies reveal a novel role for the conserved Glu residue in the establishment of mismatch discrimination by MutS.

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The RecA protein of Escherichia coli (M r 37,842, 352 amino acids) is essential for homologous genetic recombination and for the postreplicative repair of damaged DNA. The purified RecA protein will promote a variety of DNA pairing reactions that presumably reflect in vivo recombination functions. The most extensively investigated DNA pairing activity is the ATPdependent three-strand exchange reaction in which a circular ssDNA 1 molecule and a homologous linear dsDNA molecule are recombined to yield a nicked circular dsDNA molecule and a linear ssDNA molecule. This reaction proceeds in three phases. In the first phase, the circular ssDNA substrate is coated with RecA protein to form a presynaptic complex; this complex will catalyze the hydrolysis of ATP to ADP and P i . In the second phase, the presynaptic complex interacts with a dsDNA molecule, the homologous sequences are brought into register, and pairing between the circular ssDNA and the complementary strand from the dsDNA is initiated. In the third phase, the complementary linear strand is completely transferred to the circular ssDNA by unidirectional branch migration to yield the nicked circular dsDNA and displaced linear ssDNA products (1).The x-ray crystal structure of the RecA protein indicates that the phosphate groups of the nucleotide cofactor, ATP, are bound by a loop consisting of amino acids 66 through 73 (2). The sequence of this loop ( 66 GPESSGKT 73 ) corresponds to a variation of the well known phosphate binding loop (P-loop) consensus sequence (GXXXXGK[T/S]) found in many NTP-binding proteins (3). The invariant lysine and threonine/serine residues in the P-loop motif are generally found to interact directly with the ␤ and ␥ phosphates of ATP and have been shown for the RecA protein (as well as many other proteins) to be directly involved in the catalysis of phosphoryl transfer (4). Interestingly, although the four variable residues (XXXX) in this sequence can differ widely in different classes of proteins, the specific sequence, GPESSGKT, is highly conserved in over sixty different bacterial RecA proteins (1).As part of an investigation of the role of the P-loop in ATP hydrolysis, we prepared a mutant RecA protein in which the serine residue at position 69 was replaced by a glycine. The biochemical properties of the [S69G]RecA protein provide new insight into the coupling of NTP hydrolysis and DNA strand exchange and are described in this report. EXPERIMENTAL PROCEDURESMaterials-Wild type RecA protein was prepared as described previously (5). rATP was from Sigma; dATP, ddATP, [␣-32 P]rATP, [␣-32 P]dATP, and [␣-32 P]ddATP were from Amersham Pharmacia Biotech. E. coli SSB was from Promega. Circular X ssDNA ((ϩ)-strand) and circular X dsDNA were from New England Biolabs; linear X dsDNA was prepared from circular X dsDNA as described (6). Singleand double-stranded DNA concentrations were determined by absorbance at 260 nm using the conversion factors 36 and 50 g/ml/A 260 , respectively. All DNA concentrations are expressed as total nucleotides....

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