Abstract
The MutS DNA mismatch repair protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. To identify regions of MutS protein in close proximity to the heteroduplex DNA, we have utilized the photoactivated cross-linking moiety 5-iododeoxyuridine (5-IdUrd). Nucleoprotein complexes of Thermus aquaticus MutS protein bound to monosubstituted 5-IdUrd-containing heteroduplex DNAs were cross-linked with long-wavelength ultraviolet light. Positioning of the 5-IdUrd moiety at one of three positions within the DNA bulge, two nucleotides upstream or three nucleotides downstream of the unpaired base, resulted in an identical subset of cross-linked peptides as determined by proteolytic fingerprinting. The tryptic peptide cross-linked to an unpaired 5-IdUrd residue was determined by peptide sequencing to correspond to a highly conserved region spanning residues 25-49. Cross-linking to the bulge nucleotide occurred at Phe-39, indicating that this residue contacts, or is in close proximity to, the unpaired base of a heteroduplex DNA. Site-directed mutagenesis resulting in the substitution of Ala for Phe-39 reduced the affinity of the mutant protein for heteroduplex DNA by roughly 3 orders of magnitude, but had no apparent effect on its ability to dimerize, its thermostability, or its ATPase activity. These results implicate the region in the vicinity of Phe-39 as being crucial for heteroduplex DNA binding by Taq MutS protein.
Highlights
Recognition of mispaired or unpaired bases during mismatch repair is carried out by the family of MutS proteins whose members are found in many organisms from bacteria to man
Heteroduplex DNA Binding by Taq MutS Protein—The photocross-linking scheme necessitated modification of heteroduplex DNA substrates containing an unpaired base, usually a thymidine
Since the van der Waals radius of iodine is only 8% larger than that of the methyl group it replaces [6], it is unlikely that the monosubstitution of thymine by photoactive 5-IdUrd would significantly alter the structure of the MutS nucleoprotein complex
Summary
Recognition of mispaired or unpaired bases during mismatch repair is carried out by the family of MutS proteins whose members are found in many organisms from bacteria to man. To identify regions of MutS protein in close contact with the heteroduplex DNA, we have carried out photocross-linking of nucleoprotein complexes containing a MutS protein from Thermus aquaticus bound to a derivatized heteroduplex DNA containing 5-iododeoxyuridine (5-IdUrd), a photoactivated zerolength cross-linker [6]. The success of the approach hinges on the requirement for close proximity of an amino acid especially in the case of a zero-length cross-linking moiety, the need for the amino acid to assume an appropriate geometry for cross-linking, and the chemical reactivity of a potential target amino acid For these reasons, not all amino acid residues in close contact with the DNA will be identified by cross-linking. Incidental cross-linking to regions of a protein not involved in DNA binding is minimized because the 5-IdUrd
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
