Human DNA polymerase eta (Pol eta) is the gene product underlying xeroderma pigmentosum variant, and plays principal roles in translesion DNA synthesis. Here, we identified human MLH1, an essential component of mismatch repair (MMR), as a Pol eta-interacting protein. The middle area residues, which include the little finger domain, of Pol eta are important for the interaction with MLH1. Pol eta also interacts with the MLH1/PMS2 heterodimer (MutL alpha). Co-immunoprecipitation analyses revealed that MutLa, and also MSH2 and MSH6, components of the MutS alpha heterodimer, form complexes with Pol eta in human cells. Although MutSa had been reported to interact with C-terminal residues of Pol eta, MutL alpha and MutS alpha co-precipitated with C-terminally truncated Pol eta, suggesting that MutS alpha can interact with Pol eta through MutL alpha. MMR proteins were more abundant in the Pol eta complex on the chromatin of S phase-synchronized cells than of asynchronous cells, suggesting that the interaction between Pol eta and MLH1 is involved in DNA replication. (C) 2009 Elsevier Inc. All rights reserved.