In addition to their mismatch recognition activities, bacterial and eukaryotic MutS activities have an associated ATPase activity that is required for function of the proteins in mismatch repair (1-5). Two distinct functions have been proposed for nucleotide binding and hydrolysis by MutS homologs, both of which are based on the effects of ATP on MutS-heteroduplex interaction. The presence of ATP greatly reduces the efficiency of specific complex formation between bacterial MutS or eukaryotic MutS␣ and heteroduplex DNA (5-10), and ATP challenge of preformed MutS⅐heteroduplex complexes has been shown to result in departure of the protein from the mismatch (11). Available information indicates that some of these effects are attributable to ATP binding. Thus, ATP␥S has been shown to promote departure of MutS from the mismatch in heteroduplex DNA (11), while ATP␥S or ATP (in the absence of a divalent cation) reduce the binding efficiency human MutS␣ (hMutS␣) to synthetic heteroduplexes (5, 10).Electron microscopy of complexes between bacterial MutS and large heteroduplexes prepared from natural DNAs has demonstrated that ATP-promoted release of MutS from a mismatch is associated with efficient conversion of protein⅐DNA complexes to ␣-shaped loop structures stabilized by MutS at the base (11). Loop formation requires a mismatch, loop size increases linearly with time, loop growth depends on continued ATP hydrolysis, and the mismatch usually ends up in the loop. These observations have been interpreted in terms of a mechanism in which ATP binding reduces affinity of the protein for a mispair and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (11). MutS movement in this manner has been postulated to be important for the coupling of mismatch recognition to loading of the excision system at the strand break that directs repair (12, 13), a site that can be located hundreds of base pairs from this mismatch.The finding that ATP binding reduces the efficiency of specific complex formation between hMutS␣ and oligonucleotide heteroduplexes has led to proposal of a molecular switch model for action of MutS activities. Like a G-protein, hMutS␣ is postulated to exist in two states, an ADP-bound form that binds with near irreversible affinity to a mismatch and an ATPbound form that does not (10). In this proposal hMutS␣⅐ADP binds to a mispair and recruits downstream activities to this site. After assembly of the excision system, ATP binding results in dissociation of hMutS␣ from the heteroduplex so that repair may proceed (10).To further clarify the role(s) of ATP binding and hydrolysis in hMutS␣ action, we have evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of hMutS␣⅐DNA complexes and have examined the effect of DNA topology on ATP-promoted dissociation of hMutS␣ complexes with small heteroduplexes. We demonstrate that ADP is not required for mismatch recognition by hMutS␣, but...

Human replication protein A, a single-stranded DNA (ssDNA)-binding protein, is a required factor in eukaryotic DNA replication and DNA repair systems and has been suggested to function during DNA recombination. The protein is also a target of interaction for a variety of proteins that control replication, transcription, and cell growth. To understand the role of hRPA in these processes, we examined the binding of hRPA to defined ssDNA molecules. Employing gel shift assays that "titrated" the length of ssDNA, hRPA was found to form distinct multimeric complexes that could be detected by glutaraldehyde cross-linking. Within these complexes, monomers of hRPA utilized a minimum binding site size on ssDNA of 8 to 10 nucleotides (the hRPA8_10t complex) and appeared to bind ssDNA cooperatively. Intriguingly, alteration of gel shift conditions revealed the formation of a second, distinctly different complex that bound ssDNA in roughly 30-nucleotide steps (the hRPA30t complex), a complex similar to that described by Kim et al. (C. Kim, R. 0. Snyder, and M. S. Wold, Mol. Cell. Biol. 12:3050-3059, 1992). Both the hRPA8_l10t and hRPA30t complexes can coexist in solution. We speculate that the role of hRPA in DNA metabolism may be modulated through the ability of hRPA to bind ssDNA in these two modes.The replication, recombination, and repair of the cellular genetic information entail interconversion of DNA between the duplex and single-stranded forms. Study of nucleic acid enzymology has shown that single-stranded DNA (ssDNA) generated within cells is invariably associated with protein factors, often ssDNA-binding proteins (SSBs). SSBs combine with the ssDNA to form protein-DNA complexes that maintain the unwound state and are more active as substrates, for example, in DNA replication (12). A clear understanding of the architecture of SSB-ssDNA complexes is required to comprehend the function of SSB in DNA metabolism and determine how these processes are regulated within the cell.Eukaryotic SSBs with defined roles in DNA enzymology have recently been isolated, one of which, from human cells, is termed human replication protein A (hRPA). hRPA, a heterotrimer with subunits of 68, 29, and 14 kDa, was initially isolated as a factor from primate cells required to support simian virus 40 (SV40) DNA replication in vitro (24,42,43). The factor was identified as an SSB by various criteria including the high affinity of hRPA for ssDNA (24,42,43) and ability to stimulate human DNA polymerase a, an essential eukaryotic replication factor (22, 31). hRPA is phosphorylated in a cell-cycle specific manner, suggesting that DNA replication and other hRPA-mediated events may be modulated through regulation of hRPA activity (18,21,25). Moreover, recent studies have shown that hRPA specifically interacts with a number of transcription factors, suggesting that these factors regulate DNA replication, in part, through hRPA (20, 27, 35). In addition to its defined role in SV40 DNA replication, hRPA is also a required factor in an in vitro system...

Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA 8nt ) and with 30 nucleotides (hRPA 30nt ). We find the relatively unstable hRPA 8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA 30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA 30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA 30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.

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Formation of a ternary complex between human MutS␣, MutL␣, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutL␣⅐hMutS␣⅐heteroduplex complex requires a mismatch and ATP hydrolysis, and depends on DNA chain length. Ternary complex formation was supported by a 200-base pair G-T heteroduplex, a 100-base pair substrate was somewhat less effective, and a 41-base pair heteroduplex was inactive. As judged by surface plasmon resonance spectroscopy, ternary complexes produced with the 200-base pair G-T DNA contained ϳ0.8 mol of hMutL␣/mol of heteroduplex-bound hMutS␣. Although the steady-state levels of the hMutL␣⅐hMutS␣⅐ heteroduplex were substantial, this complex was found to turn over, as judged by surface plasmon resonance spectroscopy and electrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dissociation occurred in the latter case upon challenge with competitor DNA. However, ternary complex dissociation as monitored by gel shift assay was prevented if both ends of the heteroduplex were physically blocked with streptavidin⅐biotin complexes. This observation suggests that, like hMutS␣, the hMutL␣⅐hMutS␣ complex can migrate along the helix contour to dissociate at DNA ends.The MutS and MutL homologs, which are required for the initiation of mismatch repair, have been implicated in the correction of DNA biosynthetic errors, the transcription-coupled repair of DNA damage, and the fidelity of genetic recombination (1-6). In mammalian cells, MutS␣ (MSH2⅐MSH6 heterodimer), MutS␤ (MSH2⅐MSH3 heterodimer), and MutL␣ (MLH1⅐PMS2 heterodimer) are also thought to function as lesion sensors for certain types of DNA damage that kill by activating apoptosis (2, 6, 7).Repair in the Escherichia coli system is initiated by the binding of MutS to a mismatch (8 -10). Formation of a MutL⅐MutS⅐heteroduplex complex has been demonstrated by DNase I footprint analysis (11), electron microscopy (12), and surface plasmon resonance spectroscopy (SPRS) 1 (13), with assembly of this ternary complex being ATP-dependent. Several lines of evidence indicate that assembly of the ternary complex is required for subsequent steps in mismatch repair. Both MutS and MutL are required for the mismatch-dependent activation of the d(GATC) endonuclease activity of MutH, which cleaves the unmethylated strand of a hemimethylated d(GATC) site, with the ensuing strand break serving to direct repair to the unmethylated DNA strand (14). MutS and MutL are also required for the mismatch-dependent activation of DNA helicase II, which enters the helix at the strand break and initiates the excision step of repair (15). Formation of a MutL␣⅐MutS␣⅐heteroduplex complex has been demonstrated by electrophoretic gel shift analysis with yeast mismatch repair proteins using synthetic heteroduplexes of ϳ50 base pairs (bp) in size (16, 17). However, using gel shift methods and surface plasmon reson...

Analytical equilibrium ultracentrifugation indicates that Escherichia coli MutS exists as an equilibrating mixture of dimers and tetramers. The association constant for the dimer-to-tetramer transition is 2.1 ؋ 10 7 M ؊1 , indicating that the protein would consist of both dimers and tetramers at physiological concentrations. The carboxyl terminus of MutS is required for tetramer assembly because a previously described 53-amino acid carboxyl-terminal truncation (MutS800) forms a limiting species of a dimer (Obmolova, G., Ban, C., Hsieh, P., and Yang, W. MutS homologs participate in multiple genetic stabilization pathways by virtue of their ability to recognize a spectrum of DNA lesions. Recognition of base pair mismatches by MutS homologs has been implicated in the rectification of DNA biosynthetic errors and in the dissolution of recombination events involving diverged sequences (1-5). Recognition of damaged base pairs by MutS homologs is also involved in the transcription-coupled repair of DNA damage (2, 4) and has been implicated in the activation of checkpoint and apoptotic responses to certain types of DNA damage in mammalian cells (6). Members of the MutS family can thus be viewed as molecular sentinels that respond to subtle variations in DNA structure or dynamics and then communicate presence of that lesion to downstream activities (1, 7-9).Events downstream of MutS recognition have been delineated for the Escherichia coli pathway responsible for correction of DNA biosynthetic errors, and this reaction has been reconstituted in vitro using near homogeneous components. Although a number of mechanistic details remain to be established, basic roles of the individual activities have been defined. MutS recruits MutL to the heteroduplex in a reaction requiring ATP (10 -12). Assembly of this ternary complex is sufficient to activate the latent endonuclease activity of MutH, which incises the unmethylated strand at a hemimethylated d(GATC) strand signal (13). In addition, this complex activates the unwinding activity of DNA helicase II, which loads at the MutH incision with an orientation bias so that helix unwinding proceeds toward the mismatch (14, 15). The unwound portion of the incised strand is hydrolyzed by a single-strand exonuclease. If unwinding proceeds from a strand break located 5Ј to the mispair, the displaced single strand is degraded by the 5Ј-to-3Ј hydrolytic activity of ExoVII or RecJ exonuclease (16,17). When helicase unwinding initiates at a nick 3Ј to the mismatch, the unwound strand is degraded by the 3Ј-to-5Ј activity of ExoI, ExoVII,. Excision terminates at a number of sites centered about 50 base pairs beyond the mismatch, the ensuing gap is repaired by DNA polymerase III holoenzyme in the presence of single-strand binding protein, and DNA ligase restores covalent integrity to the repaired strand (18,20).In addition to its mismatch recognition activity, bacterial MutS harbors a weak ATPase (21) that is stimulated by DNA (22). Because the rate-limiting step for ATP hydrolytic turnover is se...

ATP hydrolysis by MutS homologs is required for function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is controversial. In this paper we describe a steady-state kinetic analysis of the DNA-activated ATPase of human MutS␣. Comparison of salt concentration effects on mismatch repair and mismatch-provoked excision in HeLa nuclear extracts with salt effects on the DNA-activated ATPase suggests that ATP hydrolysis by MutS␣ is involved in the rate determining step in the repair pathway. While the ATPase is activated by homoduplex and heteroduplex DNA, the half-maximal concentration for activation by heteroduplex DNA is significantly lower under physiological salt concentrations. Furthermore, at optimal salt concentration, heteroduplex DNA increases the k cat for ATP hydrolysis to a greater extent than does homoduplex DNA. We also demonstrate that the degree of ATPase activation is dependent on DNA chain length, with the k cat for hydrolysis increasing significantly with chain length of the DNA cofactor. These results are discussed in terms of the translocation

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