Abstract
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.
Highlights
Mismatch repair is a conserved genetic stabilization system that in bacteria corrects DNA replication errors and ensures the fidelity of genetic recombination [1,2,3]
To clarify the role(s) of the  clamp in mismatch repair, we have evaluated the effects of the E. coli , ␥ complex, and Pol III core on individual steps of the reconstituted E. coli methyl-directed reaction
Protein-Protein Interactions That Link Replication Machinery and the Mismatch Repair System—In view of our failure to observe any significant effects of  on MutH activation or excision steps of methyl-directed mismatch repair, we re-evaluated interactions of MutS and MutL with  clamp and with the other components of Pol III holoenzyme, using a method distinct from those employed in the previous study [12]
Summary
Proteins and DNAs—MutS [13], MutSN containing four Ala substitutions within the N-terminal  interaction motif (15QQYLRL20 to 15AAYAAL20) [12], MutL [14], MutH [15], DNA helicase II [16],  clamp [17], PK (a  clamp variant containing a 6-residue C-terminal cAMP-dependent protein kinase recognition motif) [18], ␥ complex [19], and Pol III core [20] were isolated by published methods. Exonuclease I, SSB, NheI, ClaI, XhoI, and HindIII, cAMP-dependent protein kinase were obtained from commercial sources.
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