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Title: Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

Abstract

Recent evidence suggests that human O {sup 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{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} 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:
 [1];  [2];  [1];  [1];  [3]
  1. Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106 (United States)
  2. Mass Spectrometry Center, Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195 (United States)
  3. Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106 (United States). E-mail: Kalkunte.srivenugopal@ttuhsc.edu
Publication Date:
OSTI Identifier:
20713461
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 337; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2005.09.177; PII: S0006-291X(05)02219-9; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AFFINITY; ALKYLATION; CELL CYCLE; CHEMOTHERAPY; CHROMATOGRAPHY; DNA DAMAGES; DNA REPAIR; DNA REPLICATION; HEAT-SHOCK PROTEINS; HISTONES; LARGE INTESTINE; MASS SPECTROSCOPY; NEOPLASMS; RNA PROCESSING

Citation Formats

Niture, Suryakant K., Doneanu, Catalin E., Velu, Chinavenmeni S., Bailey, Nathan I., and Srivenugopal, Kalkunte S. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry. United States: N. p., 2005. Web. doi:10.1016/j.bbrc.2005.09.177.
Niture, Suryakant K., Doneanu, Catalin E., Velu, Chinavenmeni S., Bailey, Nathan I., & Srivenugopal, Kalkunte S. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry. United States. doi:10.1016/j.bbrc.2005.09.177.
Niture, Suryakant K., Doneanu, Catalin E., Velu, Chinavenmeni S., Bailey, Nathan I., and Srivenugopal, Kalkunte S. Fri . "Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry". United States. doi:10.1016/j.bbrc.2005.09.177.
@article{osti_20713461,
title = {Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry},
author = {Niture, Suryakant K. and Doneanu, Catalin E. and Velu, Chinavenmeni S. and Bailey, Nathan I. and Srivenugopal, Kalkunte S.},
abstractNote = {Recent evidence suggests that human O {sup 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{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} 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.},
doi = {10.1016/j.bbrc.2005.09.177},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 337,
place = {United States},
year = {Fri Dec 02 00:00:00 EST 2005},
month = {Fri Dec 02 00:00:00 EST 2005}
}