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Mutational analysis of the nucleotide binding domain of the mismatch repair enzyme hMSH-2
A Whitehouse1, R Parmar, J Deeble
1Molecular Medicine Unit, St. James's University Hospital, Leeds, United Kingdom. whitehouse@stjames.leeds.ac.uk
Abstract:
The genes involved in postreplicative DNA mismatch repair are a highly conserved family of proteins. In humans, germline mutations in these genes (hMSH-2, hMLH-1, hPMS-1, and hPMS-2) have been implicated in hereditary nonpolyposis colorectal cancer (HNPCC). We have previously shown that a region of high homology between the members of this class of proteins in different species contains a type A nucleotide binding site consensus sequence which has ATPase activity and is sufficient to bind DNA containing specific mismatched residues (1). To identify residues which are necessary for this activity, we have created a range of mutants containing amino acid substitutions within the nucleotide binding domain of hMSH-2. These mutants have been expressed and assessed for ATPase activity and their ability to identify mismatch-containing DNA. Here we demonstrate that a variant protein which has the conserved residue Lys 675 within the nucleotide binding consensus sequence altered to an alanine has severely impaired ATPase activity and is unable to bind DNA containing specific mismatched residues.
Insights
DNA mismatch repair proteins are crucial for preventing cancer. A key residue (Lys 675) in hMSH-2 is essential for its ATPase activity and binding to mismatched DNA, highlighting its role in hereditary nonpolyposis colorectal cancer.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Postreplicative DNA mismatch repair genes are highly conserved and crucial for genomic stability.
- Germline mutations in human genes like hMSH-2 are linked to hereditary nonpolyposis colorectal cancer (HNPCC).
- A conserved nucleotide binding site with ATPase activity is essential for DNA mismatch recognition.
Purpose of the Study:
- To identify critical residues within the hMSH-2 nucleotide binding domain necessary for its function.
- To investigate the role of specific amino acid substitutions in ATPase activity and DNA binding.
Main Methods:
- Site-directed mutagenesis was used to create hMSH-2 variants with amino acid substitutions.
- Mutant proteins were expressed and purified.
- ATPase activity assays and DNA binding assays with mismatched DNA were performed.
Main Results:
- A specific mutant, Lys 675 to Alanine, exhibited significantly reduced ATPase activity.
- This Lys 675 Alanine mutant was unable to bind DNA containing specific mismatched residues.
- The conserved Lys 675 residue is critical for both ATPase activity and mismatch recognition by hMSH-2.
Conclusions:
- The conserved Lysine at position 675 in hMSH-2 is indispensable for its ATPase activity.
- This residue is essential for the protein's ability to recognize and bind to mismatched DNA.
- Understanding these molecular mechanisms provides insight into HNPCC pathogenesis and potential therapeutic targets.