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/apyrimidinic endonuclease (APE1), proliferating cell nuclear antigen (PCNA), and replication protein A (RPA) in HeLa nuclear extracts as shown by immunoprecipitation and Western blotting. However, binding of hMYH to DNA polymerases beta and delta was not detected. By using constructs containing different portions of hMYH fused to glutathione S-transferase, we have demonstrated 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 of hMYH that contains a conserved PCNA-binding motif binds PCNA, and subsequent amino acid substitution identified Phe-518 and Phe-519 as essential residues required for PCNA binding. RPA binds to a peptide that consists of residues 6-32 of hMYH and contains a conserved RPA-binding motif. The PCNA- and RPA-binding sites of hMYH are further confirmed by peptide and antibody titration. These results suggest that hMYH repair is a long patch base excision repair pathway.
Parker
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A
Department of Biochemistry and Molecular Biology, the University of Maryland, Baltimore, Maryland 21201, USA.
Gu
Y
Y
Mahoney
W
W
Lee
S H
SH
Singh
K K
KK
Lu
A L
AL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D017124:Conserved Sequence; D045647:DNA Glycosylases; D019951:DNA Polymerase beta / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006367:HeLa Cells; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D051716:Replication Protein A; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; D019951:DNA Polymerase beta; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase