Modulation of MutS ATP Hydrolysis by DNA Cofactors

Bjornson, Keith P.; Allen, Dwayne J.; Modrich, Paul

doi:10.1021/bi992286u

Cited by 85 publications

(115 citation statements)

References 49 publications

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“…This analysis reveals that under the conditions of our experiments, both E. coli and Taq MutS exist primarily as dimers and tetramers, with Ϸ50% dimers for E. coli MutS and Ϸ75% dimers for Taq MutS, independent of whether they are bound to DNA (data not shown). These results are consistent with previous biochemical studies that indicate that the oligomeric states of MutS are independent of DNA binding (21,37). Plots of AFM volumes of MutS vs. DNA bend angles (data not shown) show no correlation between DNA bend angles and oligomeric states of MutS-DNA complexes.…”

Section: Conformations Of Muts-dna Complexes Are Independent Of Oligo-supporting

confidence: 92%

See 1 more Smart Citation

DNA bending and unbending by MutS govern mismatch recognition and specificity

Wang

Yang²,

Schofield

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

172

233

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DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion͞deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity. D NA mismatch repair (MMR) is a highly conserved repair pathway targeting mismatched bases that arise through DNA replication errors and during homologous recombination (1-3). Inactivation of MMR genes results in a significant increase in the spontaneous mutation rate and, in humans, a predisposition to cancer (4). Escherichia coli provides the best-understood MMR system and serves as a prototype for the more complicated but homologous eukaryotic systems (5). In E. coli, the proteins MutS, MutL, and MutH are responsible for the initiation of MMR (6). MutS and MutL function as dimers and have intrinsic ATPase activities that are essential for MMR (7,8). MMR is initiated by the binding of MutS to either a mismatch or a short insertion͞deletion loop (IDL). Subsequently, ATP induces a conformational change in the MutS-mismatch complex and promotes its interaction with MutL. Assembly of the MutS-MutL-heteroduplex complex activates the endonuclease activity of MutH, which incises the newly synthesized (unmethylated) strand at a d(GATC) site. This incision confers strand specificity of MMR, directing repair exclusively to the newly synthesized strand containing the error. Excision repair completes the process.Crystal structures of E. coli and Thermus aquaticus (Taq) MutS dimers complexed with a G͞T base-base mismatch and a 1T-bulge, respectively, shed light on the structural components of mismatch recognition (9-11). Specific interactions include an aromatic ring stack of a conserved phenylalanine (Phe-39 in Taq or Phe-36 in...

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Section: Conformations Of Muts-dna Complexes Are Independent Of Oligo-supporting

confidence: 92%

“…Both E. coli and Taq MutS proteins have been shown to exist in an equilibrium of dimers and tetramers (21,37,38). This observation raises the question of whether the bent and unbent populations correlate with the different oligomeric states of MutS proteins.…”

Section: Conformations Of Muts-dna Complexes Are Independent Of Oligo-mentioning

confidence: 99%

DNA bending and unbending by MutS govern mismatch recognition and specificity

Wang

Yang²,

Schofield

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

172

233

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“…The ATPase activity of Msh2, which interacts only in a sequence nonspecific manner with the DNA, appears not to be affected. Similar findings have been reported for T. aquaticus MutS, in which interaction with mismatched DNA suppresses the rapid ATP hydrolysis activity of S 1 (30-fold) but does not appear to affect the activity of S 2 ; of course in this case it was not known which subunit, S 1 or S 2 , made specific contacts with the mismatch [18]; mismatched DNA inhibits rapid ATP hydrolysis catalyzed by E. coli MutS as well, although the stoichiometry of the reaction is not resolved [35]. We know now that Msh6 in S. cerevisiae Msh2-Msh6 is the equivalent of S 1 in T. aquaticus MutS, and there exists a strong "cis" linkage between the mismatch recognition and ATPase activities Antony et al Page 10 of this subunit.…”

Section: Discussionmentioning

confidence: 96%

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

et al. 2006

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Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2-Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S 1 ) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S 2 ) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S 1 -but which of these subunits, S 1 or S 2 , makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2-Msh6 heterodimer (in Msh2-Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S 1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S 2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a "cis" linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2-Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.

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“…An important earlier study of E. coli MutS provided the first evidence for suppression of burst ATPase kinetics by mismatch recognition proteins in the presence of mismatched DNA (although the burst amplitude in the absence of DNA was extremely low-1/15th the concentration of MutS in the assay) (31). The report also did not clarify whether the rate of ATP binding or ATP hydrolysis was suppressed; therefore, it is not clear whether the MutS·mismatched DNA complex is stabilized in an ATP-free or ATP-bound state.…”

Section: Mismatched Base Pair or Single Base Insertion Modulates Atp mentioning

confidence: 99%

Mismatch Recognition-Coupled Stabilization of Msh2-Msh6 in an ATP-Bound State at the Initiation of DNA Repair

2003

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Mismatch repair proteins correct errors in DNA via an ATP-driven process. In eukaryotes, the Msh2-Msh6 complex recognizes base pair mismatches and small insertion/deletions in DNA and initiates repair. Both Msh2 and Msh6 proteins contain Walker ATP-binding motifs that are necessary for repair activity. To understand how these proteins couple ATP binding and hydrolysis to DNA binding/mismatch recognition, the ATPase activity of Saccharomyces cerevisiae Msh2-Msh6 was examined under presteady-state conditions. Acid-quench experiments revealed that in the absence of DNA, Msh2-Msh6 hydrolyzes ATP rapidly (burst rate = 3 s −1 at 20 °C) and then undergoes a slow step in the pathway that limits catalytic turnover (k cat = 0.1 s −1 ). ATP is hydrolyzed similarly in the presence of fully matched duplex DNA; however, in the presence of a G:T mismatch or +T insertioncontaining DNA, ATP hydrolysis is severely suppressed (rate = 0.1 s −1 ). Pulse-chase experiments revealed that Msh2-Msh6 binds ATP rapidly in the absence or in the presence of DNA (rate = 0.1 μM −1 s −1 ), indicating that for the Msh2-Msh6·mismatched DNA complex, a step after ATP binding but before or at ATP hydrolysis is the rate-limiting step in the pathway. Thus, mismatch recognition is coupled to a dramatic increase in the residence time of ATP on Msh2-Msh6. This mismatchinduced, stable ATP-bound state of Msh2-Msh6 likely signals downstream events in the repair pathway.Replicative DNA polymerases are responsible for accurately reproducing the genetic code of organisms; however, even the most accurate polymerases make errors that result in approximately one mismatched base pair per 10 7 nucleotides as well as insertion/deletion loops (1). These defects must be corrected prior to subsequent rounds of replication, to minimize accumulation of potentially deleterious mutations and genome instability. A multi-protein mismatch repair system is responsible for this task, which involves recognition and removal of the defects followed by replacement with correct DNA. This repair system was initially identified and studied extensively in bacteria, and homologous systems were discovered in a variety of other organisms, including humans (2). The critical role of DNA mismatch repair in maintaining genome and cellular integrity is highlighted by the links between defective repair protein function and predisposition to cancer (e.g., hereditary nonpolyposis colorectal cancer (3)).The repair process begins with DNA binding and mismatch/defect recognition by prokaryotic MutS or eukaryotic MutS homologue proteins. MutS/Msh 1 proteins then signal other proteins downstream in the pathway to initiate DNA excision. In bacteria, MutH endonuclease nicks the unmethylated new DNA strand at a GATC site, which is followed by UvrD/Helicase II and † This work was supported by a grant from the N.I.H. .

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Modulation of MutS ATP Hydrolysis by DNA Cofactors

Cited by 85 publications

References 49 publications

DNA bending and unbending by MutS govern mismatch recognition and specificity

DNA bending and unbending by MutS govern mismatch recognition and specificity

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

Mismatch Recognition-Coupled Stabilization of Msh2-Msh6 in an ATP-Bound State at the Initiation of DNA Repair

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