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Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein
Published on: March 31, 2010
The MutL ATPase is required for mismatch repair
1Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
Members of the MutL family contain a novel nucleotide binding motif near their amino terminus, and the Escherichia coli protein has been found to be a weak ATPase (Ban, C., and Yang, W. (1998) Cell 95, 541-552). Genetic analysis has indicated that substitution of Lys for Glu-32 within this motif of bacterial MutL results in a strong dominant negative phenotype (Aronshtam, A., and Marinus, M. G. (1996) Nucleic Acids Res. 24, 2498-2504). By in vitro comparison of MutL-E32K with the wild type protein, we show the mutant protein to be defective in DNA-activated ATP hydrolysis, as well as MutS- and MutL-dependent activation of the MutH d(GATC) endonuclease and the mismatch repair excision system. MutL-E32K also acts in dominant negative manner in the presence of wild type MutL in vitro, inhibiting the overall mismatch repair reaction, as well as MutH activation. As judged by protein affinity chromatography, MutL and MutL-E32K both support formation of ternary complexes that also contain MutS and MutH or MutS and DNA helicase II. These findings imply that the MutL nucleotide binding center is required for mismatch repair and suggest that the dominant negative behavior of the MutL-E32K mutation is due to the formation of dead-end complexes in which the MutL-E32K protein is unable to transduce a signal from MutS that otherwise results in mismatch-dependent activation of the MutH d(GATC) endonuclease or the unwinding activity of helicase II.
Insights
The MutL-E32K mutation impairs DNA mismatch repair by disrupting ATP hydrolysis and protein interactions. This mutant protein forms non-functional complexes, inhibiting the repair process and acting in a dominant negative manner.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The MutL family of proteins is crucial for DNA mismatch repair.
- MutL contains a nucleotide-binding motif essential for its function.
- A specific mutation, MutL-E32K, in Escherichia coli MutL causes a dominant negative phenotype.
Purpose of the Study:
- To investigate the functional consequences of the MutL-E32K mutation in vitro.
- To elucidate the mechanism underlying the dominant negative effect of MutL-E32K.
- To determine the role of the MutL nucleotide-binding center in DNA mismatch repair.
Main Methods:
- In vitro biochemical assays comparing wild-type MutL and MutL-E32K.
- Analysis of DNA-activated ATP hydrolysis.
- Assessment of MutS- and MutL-dependent activation of MutH endonuclease and mismatch repair excision.
- Protein affinity chromatography to study complex formation.
Main Results:
- MutL-E32K is defective in DNA-activated ATP hydrolysis.
- The mutant protein fails to activate MutH endonuclease and the excision system.
- MutL-E32K inhibits wild-type MutL function in vitro, demonstrating dominant negative activity.
- Both MutL and MutL-E32K form ternary complexes with MutS and MutH or helicase II.
Conclusions:
- The MutL nucleotide-binding center is essential for DNA mismatch repair.
- The dominant negative effect of MutL-E32K results from the formation of non-productive dead-end complexes.
- These complexes prevent signal transduction from MutS, leading to inhibition of MutH activation and helicase II unwinding.
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