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Role of MutS ATPase activity in MutS,L-dependent block of in vitro strand transfer
1Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, P.O. Box 12233, Research Triangle, North Carolina 27709, USA. worth@nieh.nih.gov
Abstract:
In addition to mismatch recognition, Escherichia coli MutS has an associated ATPase activity that is fundamental to repair. Hence, we have characterized two MutS mutant gene products to define the role of ATP hydrolysis in homeologous recombination. These mutants, denoted MutS501 and MutS506, have single point mutations within the Walker A motif, and rate constants for ATP hydrolysis are down 60-100-fold as compared with wild type. Both MutS501 and MutS506 retain mismatch binding and, unlike wild type, fail to relinquish this specificity in the presence of ATP, adenosine 5'-O-(thiotriphosphate), and adenosine 5'-(beta, gamma-imino)triphosphate. Both MutS501 and MutS506 blocked the level of strand transfer between M13 and fd DNAs. The level of inhibition varied between the mutants and corresponded with the relative affinities to a G/T mispair. Neither MutS501 nor MutS506, however, would afford complete block of full-length heteroduplex in the presence of MutL. DNase I footprinting data are consistent with these results, as the region of protection by MutS501 and MutS506 was unchanged in the presence of ATP and MutL. Taken together, these studies suggest that 1) MutS impedes RecA-mediated homeologous exchange as a distinct mismatch-provoked event and 2) the role of MutL is coupled to MutS-dependent ATP hydrolysis. These observations are in good agreement with the present model for E. coli methyl-directed mismatch repair.
Insights
MutS ATPase activity is crucial for DNA repair. Mutants defective in ATP hydrolysis still bind mismatches but fail to release them, impacting homeologous recombination and suggesting MutL
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Escherichia coli MutS protein possesses ATPase activity essential for DNA repair.
- Understanding the role of ATP hydrolysis in homeologous recombination is critical.
Purpose of the Study:
- To characterize two MutS mutant gene products (MutS501 and MutS506) with impaired ATPase activity.
- To define the specific role of ATP hydrolysis by MutS in homeologous recombination.
Main Methods:
- Site-directed mutagenesis to create Walker A motif mutants (MutS501, MutS506).
- Assays for ATPase activity, mismatch binding, and DNA strand transfer inhibition.
- DNase I footprinting to analyze protein-DNA interactions.
Main Results:
- MutS501 and MutS506 exhibited 60-100 fold reduced ATP hydrolysis rates compared to wild type.
- Mutants retained mismatch binding but failed to release it in the presence of ATP analogs.
- Mutants inhibited M13/fd DNA strand transfer, with inhibition correlating to G/T mispair affinity.
- MutL did not fully block heteroduplex formation with MutS mutants, and DNase I footprints were unaffected by ATP/MutL.
Conclusions:
- MutS hinders RecA-mediated homeologous exchange as a mismatch-dependent event.
- MutL's function is linked to MutS-dependent ATP hydrolysis.
- Findings align with the established model for E. coli methyl-directed mismatch repair.