MutS and MutL Activate DNA Helicase II in a Mismatch-dependent Manner

Abstract

MutS, MutL, and DNA helicase II are required for the mismatch-provoked excision step that occurs during Escherichia coli methyl-directed mismatch repair. In this study MutL is shown to enhance the unwinding activity of DNA helicase II more than 10-fold on a conventional helicase substrate in which a 35-residue oligonucleotide is annealed to a M13 circular single-stranded phage DNA under conditions where the two proteins are present at approximately molar stoichiometry with respect to the substrate. MutS-and MutL-dependent activation of DNA helicase II has also been demonstrated with a model substrate in which a 138-residue oligonucleotide was hybridized to a 138-nucleotide gap in an otherwise duplex 7,100-base pair circular DNA. Displacement of the oligonucleotide requires MutS, MutL, DNA helicase II, and ATP and is dependent on the presence of a mismatch within the hybrid region. Although DNA helicase II and Rep helicase share substantial sequence homology and features of mechanism, Rep helicase is inactive in this reaction.Escherichia coli methyl-directed mismatch repair initiates via the mismatch-provoked incision of the unmethylated strand at a hemimethylated d(GATC) sequence in a reaction that involves the MutS-and MutL-dependent activation of the MutH d(GATC) endonuclease activity (1). The single-strand break thus produced may occur either 3Ј or 5Ј to the mismatch on the unmethylated strand and directs the exonucleolytic excision of that portion of the unmodified strand spanning the incised d(GATC) sequence and the mispair (2, 3). Excision requires MutS, MutL, DNA helicase II, and an appropriate exonuclease. When the strand break that directs repair occurs 5Ј to the mismatch, excision requires RecJ exonuclease or exonuclease VII (3, 4), both of which support 5Ј 3 3Ј hydrolysis (5, 6). For repair directed by a strand break 3Ј to the mismatch, the 3Ј 3 5Ј hydrolytic activity of exonuclease I (7) is sufficient to meet the exonuclease requirement (2). 1Since helicase II is required for excision from either side of the mismatch and because each of these exonucleases is specific for single-stranded DNA (5-7), the action of helicase II presumably serves to unwind the incised strand so as to render it exonuclease sensitive. According to this interpretation, the exonuclease functions in excision are secondary to those of DNA helicase II. We have therefore sought partial reactions in which MutS, MutL, and a mismatch might enhance the activity of helicase II. We show here that MutL stimulates helicase II activity on a conventional substrate and that helicase activity on incised duplex DNA is enhanced by MutS and MutL in a mismatch-dependent manner. The accompanying paper (8) demonstrates that MutS, MutL, and mismatch-dependent entry of helicase II into an incised heteroduplex occurs at the strand break with helicase entry biased so that translocation occurs toward the mispair. EXPERIMENTAL PROCEDURESProteins, DNA, and Nucleotides-MutS (9) and DNA helicase II (10) were purified as described. Rep helicase (11) was kind...

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

“…MutL also interacts with MutS (9) and with UvrD (14,15). Taken together, these results support the previously proposed hypothesis that MutL acts to bring together other protein components of the mismatch repair pathway (15,22,23). Interestingly, MutL has been demonstrated to stimulate the helicase activity of UvrD (14,15), the rate of MutS-mediated DNA loop formation at the site of a mismatched base (24), and in this report, the endonuclease activity of MutH.…”

“…MutL also interacts with MutS (9) and with UvrD (14,15). Taken together, these results support the previously proposed hypothesis that MutL acts to bring together other protein components of the mismatch repair pathway (15,22,23).…”

“…However, this activity is markedly stimulated in the presence of MutS, MutL, ATP, Mg 2ϩ , and a DNA substrate containing a mispaired base (4). MutL is known to stimulate the helicase activity of UvrD (15), with which it physically interacts (14). In an effort to identify the functional role of the interaction between MutL and MutH, we examined the effect of MutL on the endonuclease activity of MutH in the absence of MutS, ATP, and/or a mismatch-containing DNA substrate.…”

“…Simultaneously, a biochemical interaction was reported between MutL and UvrD (15). To identify other potential interactions involving E. coli mismatch repair proteins, all possible pairwise combinations of MutS, MutL, MutH, and UvrD were tested for interactions using the yeast twohybrid system.…”

Human MutLα: The jack of all trades in MMR is also an endonuclease

“…The preference for a discontinuous over continuous strand is unprecedented. In E. coli, after MutH makes a nick in the daughter strand, the UvrD helicase and an exonuclease follow the daughter strand from the nick towards the mismatch site in single nucleotide steps [3,8,25,26]. Kadyrov et al showed that nicking by MutLα precedes daughter strand removal by ExoI [4].…”