Functions of MutLα, Replication Protein A (RPA), and HMGB1 in 5′-Directed Mismatch Repair

A purified system comprised of MutS alpha, MutL alpha, exonuclease 1 (Exo1), and replication protein A(RPA)(in the absence or presence of HMGB1) supports 5'-directed mismatch-provoked excision that terminates after mismatch removal. MutL alpha is not essential for this reaction but enhances excision termination, although the basis of this effect has been uncertain. One model attributes the primary termination function in this system to RPA, with MutL alpha functioning in a secondary capacity by suppressing Exo1 hydrolysis of mismatch-free DNA (Genschel, J., and Modrich, P. (2003) Mol. Cell 12, 1077-1086). A second invokes MutL alpha as the primary effector of excision termination (Zhang, Y., Yuan, F., Presnell, S. R., Tian, K., Gao, Y., Tomkinson, A. E., Gu, L., and Li, G. M. (2005) Cell 122, 693-705). In the latter model, RPA provides a secondary termination function, but together with HMGB1, also participates in earlier steps of the reaction. To distinguish between these models, we have reanalyzed the functions of MutL alpha, RPA, and HMGB1 in 5'-directed mismatch-provoked excision using purified components as well as mammalian cell extracts. Analysis of extracts derived from A2780/AD cells, which are devoid of MutL alpha but nevertheless support 5'-directed mismatch repair, has demonstrated that 5'-directed excision terminates normally in the absence of MutL alpha. Experiments using purified components confirm a primary role for RPA in terminating excision by MutS alpha-activated Exo1 but are in consistent with direct participation of MutL alpha in this process. While HMGB1 attenuates excision by activated Exo1, this effect is distinct from that mediated by RPA. Assay of extracts derived from HMGB1(+/+) and HMGB1(-/) mouse embryo fibroblast cells indicates that HMGB1 is not essential for mismatch repair.