title: Proteomic analysis of human O6-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry.
abstract: Recent evidence suggests that human O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase delta, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21(waf1/cip1)), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1alpha), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90alpha and beta, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
authors.forename: Suryakant K
authors.initials: SK
authors.lastname: Niture
authors.forename: Catalin E
authors.initials: CE
authors.lastname: Doneanu
authors.forename: Chinavenmeni S
authors.initials: CS
authors.lastname: Velu
authors.forename: Nathan I
authors.initials: NI
authors.lastname: Bailey
authors.forename: Kalkunte S
authors.initials: KS
authors.lastname: Srivenugopal
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D004247:DNA / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D006657:Histones / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D040901:Proteomics
chemical_list: D006657:Histones; D004247:DNA; D019853:O(6)-Methylguanine-DNA Methyltransferase