Mutations in the MutSa interaction interface of MLH1 can abolish DNA mismatch repair Guido Plotz 1, *, Christoph Welsch 1,2 , Luis Giron-Monzon 3 , Peter Friedhoff 3 , Mario Albrecht 2 , Albrecht Piiper 1 , Ricardo M. Biondi 1 , Thomas Lengauer 2 , Stefan Zeuzem 1 and Jochen Raedle 1 1 Klinik fu ¨r Innere Medizin II, Geba ¨ ude 41, Kirrberger Straße, Universita ¨t des Saarlandes, D-66421 Homburg/Saar, Germany, 2 Max Planck Institut fu ¨ r Informatik, Stuhlsatzenhausweg 85, D-66123 Saarbru ¨ cken, Germany and 3 Institut fu ¨ r Biochemie (FB 08), Justus-Liebig-Universita ¨ t Giessen, D-35392 Giessen, Germany Received June 29, 2006; Revised October 10, 2006; Accepted October 20, 2006 ABSTRACT MutLa, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detec- tor MutSa and assembles and controls further repair enzymes. We tested if the interaction of MutLa with DNA-bound MutSa is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation inter- fered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small acces- sible surface patch, suggesting that the major interaction interface of MutLa for MutSa is located on the edge of an extensive b-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a con- served potential protein–protein interaction inter- face which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site- specifically crosslinked to MutS from this patch, confirming that the bacterial MutL–MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLa–MutSa interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development. INTRODUCTION The activity of the mismatch repair system elevates replication fidelity by several hundredfold through the removal of a wide variety of polymerase errors, including insertion–deletion loops that can form during the replication of repetitive sequences (1–3). The system has been conserved throughout evolution. In humans, germline mutations in mismatch repair genes, predominantly MLH1 and MSH2, underlie the Lynch syndrome (also called hereditary non-polyposis colorectal cancer, HNPCC), a hereditary cancer predisposition which accounts for 3-5% of all colorectal cancer cases (3–5). Mismatch repair in humans is initiated by one of two MutS heterodimers, either MutSa (MSH2–MSH6) or MutSb (MSH2–MSH3), depending on the type of mismatch to be repaired (6-8). After mismatch binding by this heterodimer, a MutL heterodimer is recruited. This is predominantly MutLa (MLH1–PMS2), although a contribution of MutLg (MLH1–MLH3) has also recently been reported for a subset of replication errors (9-11). The human MutSa/b and MutLa/ g heterodimers have evolved from the homodimeric bacterial predecessors MutS and MutL. The heterodimeric subunits of the human proteins share a common architecture, but diverge in structural and functional details. Together with a MutS protein, the MutL protein directs the exonucleolytic degradation of a stretch of the error- containing strand including the mismatched base (12–17). Repair is completed by DNA re-synthesis on the emerging gap. The overall mechanism of mismatch repair appears to be similar in all organisms except for identification of the faulty DNA strand (that has to be repaired), which is performed in E.coli and some other bacteria by MutH, an endonuclease that binds in a site-directed manner to the tran- siently hemimethylated DNA that arises during bacterial replication (18,19). Since eukaryotes lack this transient hemimethylation, other ways of strand discrimination are possible [reviewed in (1,2)]. While the role of MutS proteins as mismatch-detectors is well established, the contribution of MutL proteins to repair has remained more elusive. Recently, Modrich and co-workers have demonstrated an endonucleoylic activity of human MutLa residing in the C-terminal domain of PMS2 (20). Functionally, MutL proteins have been shown to confer termination of the exonucleolytic degradation of the faulty *To whom correspondence should be addressed. Tel: +49 6841 16 23253; Fax: +49 6841 16 23570; Email: guido.plotz@uniklinik-saarland.de Ó 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 6574–6586 Nucleic Acids Research, 2006, Vol. 34, No. 22 Published online 28 November 2006 doi:10.1093/nar/gkl944 by guest on January 14, 2015 http://nar.oxfordjournals.org/ Downloaded from