Involvement of the β Clamp in Methyl-directed Mismatch Repair in Vitro

• Published in: The Journal of biological chemistry 2009-11, Vol.284 (47), p.32782-32791
• Main Authors: Pluciennik, Anna, Burdett, Vickers, Lukianova, Olga, O'Donnell, Mike, Modrich, Paul
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Base Pair Mismatch; Blotting, Western; Catalytic Domain; DNA Polymerase III - genetics; DNA Repair; DNA Repair Enzymes - metabolism; DNA, Superhelical; DNA-Binding Proteins - metabolism; DNA: , Repair, Recombination, and Chromosome Dynamics; Endodeoxyribonucleases - metabolism; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; In Vitro Techniques; Mutation; MutL Proteins; MutS DNA Mismatch-Binding Protein - metabolism; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.054528

We have examined function of the bacterial β replication clamp in the different steps of methyl-directed DNA mismatch repair. The mismatch-, MutS-, and MutL-dependent activation of MutH is unaffected by the presence or orientation of loaded β clamp on either 3′ or 5′ heteroduplexes. Similarly, β is not required for 3′ or 5′ mismatch-provoked excision when scored in the presence of γ complex or in the presence of γ complex and DNA polymerase III core components. However, mismatch repair does not occur in the absence of β, an effect we attribute to a requirement for the clamp in the repair DNA synthesis step of the reaction. We have confirmed previous findings that β clamp interacts specifically with MutS and MutL (López de Saro, F. J., Marinus, M. G., Modrich, P., and O'Donnell, M. (2006) J. Biol. Chem. 281, 14340–14349) and show that the mutator phenotype conferred by amino acid substitution within the MutS N-terminal β-interaction motif is the probable result of instability coupled with reduced activity in multiple steps of the repair reaction. In addition, we have found that the DNA polymerase III α catalytic subunit interacts strongly and specifically with both MutS and MutL. Because interactions of polymerase III holoenzyme components with MutS and MutL appear to be of limited import during the initiation and excision steps of mismatch correction, we suggest that their significance might lie in the control of replication fork events in response to the sensing of DNA lesions by the repair system.