We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI copurifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5 to 3 hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5 or 3 to the mispair. When the strand break that directs repair is located 3 to the mispair, hEXOI-and mismatch-dependent gap formation in excision-depleted extracts requires both hMutS␣ and hMutL␣. However, excision directed by a 5 strand break requires hMutS␣ but can occur in absence of hMutL␣. In systems comprised of pure components, the 5 to 3 hydrolytic activity of hEXOI is activated by hMutS␣ in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5-heteroduplex correction. The involvement of hEXOI in 3-heteroduplex repair suggests that it has a regulatory/ structural role in assembly of the 3-excision complex or that the protein possesses a cryptic 3 to 5 hydrolytic activity.Human cells possess a strand-specific mismatch repair system that is similar to that of Escherichia coli and depends on structural and functional homologs of bacterial MutS and MutL (1-4). Inactivation of genes that encode the mammalian MutS homologs MSH2 or MSH6 or the MutL homologs MLH1 or PMS2 confers genetic instability and a predisposition to tumor development. Eleven activities have been implicated in E. coli methyl-directed mismatch repair, and the reaction has been reconstituted in a pure system (5-8). However, our understanding of the reaction in higher cells is limited.Analysis of the human reaction in nuclear extracts, using model heteroduplexes in which a strand-specific single strand break directs repair to the incised DNA strand (9, 10), has indicated that the reaction occurs in several steps by a mechanism similar to that of E. coli mismatch correction (11,12). Repair is initiated via mismatch recognition by hMutS␣ (the hMSH2⅐hMSH6 heterodimer) (13,14) or hMutS␤ (hMSH2⅐ hMSH3 heterodimer) (15-17). hMutL␣ (hMLH1⅐hPMS2 heterodimer) and PCNA 1 are also required during the earliest stages of the reaction since inactivation of either of these activities blocks repair at or prior to initiation of excision (18 -20), which removes that portion of the incised strand spanning the strand break and the mispair (21,22). Subsequent repair DNA synthesis depends on DNA polymerase ␦ and PCNA (23, 24). hRPA, the human single-stranded DNA binding protein, has also been implicated in mismatch repair (25), but the stages of the reaction during which this protein functions have not been defined.The excision step of E. coli methyl-directed mismatch correction depends on DNA helicase II, as well as several 3Ј to 5Ј and 5Ј to 3Ј exonucleases that display specificity for single-stranded DNA (6 -8, 26). Several activities have b...

Homologous pairing and strand exchange, which are catalyzed by Escherichia coli RecA protein, are central to homologous recombination. Homologs of this protein are found in eukaryotes; however, little has been reported on the recombinase activities of the mammalian homologs, including the human protein, denoted HsRad51. For the studies described here, we purified HsRad51 from E. coli. Although the activities of HsRad51 and RecA were qualitatively similar in the presence of ATP, there were also striking differences. The stoichiometry of binding to DNA and the rate of renaturation of complementary strands were similar for the two proteins, but rates of ATP hydrolysis, homologous pairing, and subsequent strand exchange promoted by HsRad51 were less than 1 ⁄10 those of RecA. In addition, HsRad51 bound ␥-thio-ATP and formed stable presynaptic complexes that promoted renaturation as rapidly as RecA, but the recombinant human protein catalyzed neither strand exchange nor homologous pairing of a single strand with duplex DNA in the presence of the ATP analog. By contrast, RecA promoted both of the latter reactions in control experiments. These observations suggest that among RecA-like proteins, HsRad51 may be a variant in which homologous pairing and strand exchange are more closely linked to the hydrolysis of ATP.

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. In this study, we measured RecA-catalyzed pairing and strand exchange in solution by energy transfer between fluorescent dyes on the ends of deoxyribo-oligonucleotides. By varying the position of the dyes in separate assays, we were able to detect the pairing of single-stranded RecA filament with duplex DNA as an increase in energy transfer, and strand displacement as a decrease in energy transfer. With these assays, the kinetics of pairing and strand displacement were studied by stopped-flow spectrofluorometry. The data revealed a rapid, second order, reversible pairing step that was followed by a slower, reversible, first order strand exchange step. These data indicate that an initial unstable intermediate exists which can readily return to reactants, and that a further, rate-limiting step (or steps) is required to effect or complete strand exchange.RecA is a 38-kilodalton protein from Escherichia coli, which has been shown to be necessary for conjugal homologous recombination in vivo (1). In vitro, RecA protein polymerizes on single-stranded DNA in the 5Ј to 3Ј direction to form a righthanded helical structure in which the DNA is extended to 1.5 times its original length (2, 3). Pairing with homologous duplex DNA results in a rapid uptake of the double-stranded DNA into a three-stranded complex, which can be kilobases in length (4 -6). Strand exchange results in displacement of the strand of duplex DNA that has the same sequence as the filament strand; the strand is displaced in the 5Ј to 3Ј direction (7-9). Homologs of RecA exist in eukaryotes from yeast to man and have been found to hydrolyze ATP, to form nucleoprotein filaments, to pair homologous DNA, and to carry out strand exchange in ways that are qualitatively similar to RecA protein (10 -15). Studies of the mechanisms of E. coli RecA may help to shed light on eukaryotic as well as prokaryotic recombination.Most studies on the kinetics of RecA-catalyzed strand exchange have used phage DNA that is several kilobases in length (16 -20). RecA filaments formed on long single-stranded DNA generate coaggregates with duplex DNA that concentrate the DNA but also limit diffusion (21). Despite this complication, joint molecule formation displayed the saturation of rates with increasing substrate concentration that is typical of MichaelisMenten kinetics (17). This observation suggested the existence of a reversible pairing step and a second, rate-limiting step in the reaction. Yancey-Wrona and Camerini-Otero (22) developed a solution assay for pairing and stable synapsis of a single-stranded oligonucleotide with duplex DNA in which a RecA filament formed in the presence of ATP␥S 1 protects a restriction site in the duplex target molecule. With this assay, they found that pairing was second order, reversible, and independent of the complexity of the target. They were...