Human homolog of the MutY repair protein (hMYH) physically interacts with proteins involved in long patch DNA base excision repair

The human MutY homolog (hMYH) is a DNA glycosylase involved in the removal of adenines or 2-hydroxyadenines misincorporated with template guanines or 7,8-dihydro-8-oxodeoxyguanines. hMYH is associated in vivo with apurinic/ap yrimidinic endonuclease (APE1), proliferating cell nuclear antigen (PCNA), and replication protein A (RPA) in HeLa nuclear extracts as shown by immuno precipitation and Western blotting. However, binding of hMYH to DNA polymer ases beta and delta was not detected. By using constructs containing differ ent portions of hMYH fused to glutathione S-transferase, we have demonstrat ed that the APE1-binding site is at a region around amino acid residue 300, that the PCNA binding activity is located at the C terminus, and that RPA binds to the N terminus of hMYH. A peptide consisting of residues 505-527 o f hMYH that contains a conserved PCNA-binding motif binds PCNA, and subsequ ent amino acid substitution identified Phe-518 and Phe-519 as essential res idues required for PCNA binding. RPA binds to a peptide that consists of re sidues 6-32 of hMYH and contains a conserved RPA-binding motif. The PCNA- a nd RPA-binding sites of hMYH are further confirmed by peptide and antibody titration. These results suggest that hMYH repair is a long patch base exci sion repair pathway.