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Research Topics
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Genetic Studies
Project Summary

Title: The Contribution of FEN-1 to Genetic Integrity Subsequent to Oxidative Stress
Synopsis: The purpose of this study is to determine the effects of oxidative stressors in mice with a certain genetic (FEN-1) deficiency.
Overall Project Objective: To test the hypothesis that a reduction in Fen-1 activity will increase the severity of the biological consequences of oxidative DNA damage. Data supporting the hypothesis will be consistent with Fen-1 having a significant role in protecting cells from the effects of oxidative DNA damage.
Status/Results to Date: The original hypothesis to be tested was that a reduction in Fen-1 activity would increase the severity of the biological consequences of oxidative DNA damage. We proposed to make and characterize the Fen-1 knockout mice prior to determining the effect of reduced Fen-1 after challenge by an oxidative stressor. However, in late 1999 and early 2000, two independent laboratories developed mice deficient in Ogg1 (the DNA glycosylase that recognizes and removes oxidized purines from DNA) and these mice were shown to have elevated spontaneous levels of oxidative base damage in DNA and elevated spontaneous mutation frequencies. Therefore, the Ogg1 null mice have been substituted for the Fen-1 knockout mice originally proposed since they are susceptible to increased spontaneous oxidative stress and were made available to us. We now have Ogg1 null mice on site and have had two litters born. The colony will be expanded to accommodate the studies using ionizing radiation and paraquat. We have also developed a quantitative in vitro base excision repair assay that has allowed us to measure the base excision repair activity in various tissues (Intano et al., 2001). By using this assay we have shown that the activity is substantially lower in somatic divisions have greater activity than post-mitotic tissues. However, the amount of the cell division among spermatogenic cells does not appear to account for the high activity observed in the cells. This assay will be directly utilized in the current project to study the effect of the Ogg1 deficiency in the response to environmental agents. We have also generated cultured cells overexposing the bacterial equivalent of Ogg1, and observed a lower spontaneous mutation frequency. These data will be compared to those obtained from Ogg1 deficient mice treated with ionizing radiation.
Project:VA-65B
Agency:Department Of Veterans' Affairs
Location:San Antonio Environmental
P.I. Name:Christi A Walter, Ph. D.
Research Type:Mechanistic
Research Focus:Environmental Toxicology
Focus Category:Genetic Studies
Status:Ongoing
Study Start Date:January 07,2000
Estimated Completion Date:January 07,2005
Specific Aims: 1) To generate embryonic stem (ES) cells carrying a targeted recombination event that results in the inactivation of one allele of murine flap endonuclease-1 (Fen-1). (2) To generate and characterize mice deficient in Fen-1. (3) To determine the effects of model oxidative stressors in mice deficient in Fen-1.
Methodology: Flap endonuclease-1 (FEN-1) has been implicated as a component of base excision repair, nonhomologous end joining and mismatch repair. Accordingly, it is logical to suggest that FEN-1 may have a central role in counteracting oxidative DNA damage.
Most Recent Publications:

Walter CA, Sung HM, Intano GW, Walter RB. Characterization of O(6)-methylguanine-DNA-methyltransferase (O(6)-MGMT) activity in Xiphophorus fishes. Mutat Res, 27;493(1-2):11-22, Jun 2001. Abstract

Walter CA, Zhou ZQ, Manguino D, Reddick R, Ikeno Y, Nelson RA, Intano GW, Herbert DC, McMahan CA, Hanes M. Health span and life span in transgenic mice with modulated DNA repair. Ann N Y Acad Sci, 928:132-40, Apr 2001. Abstract

Intano GW, McMahan CA, Walter RB, McCarrey JR, Walter CA. Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity. Nucleic Acids Res, 15;29(6):1366-72, Mar 2001. Abstract

Walter RB, Sung HM, Mitchell DL, Intano GW, Walter CA. Relative base excision repair activity in Xiphophorous fish tissue extracts. N/A, 2001. Article

Zhou ZQ, Manguino D, Kewitt K, Intano GW, McMahan CA, Herbert DC, Hanes M, Ikeno Y, Reddick R, Walter CA. Spontaneous hepatocellular carcinoma is reduced in transgenic mice overexpressing human O6- methylguanine-DNA methyltransferase. Pro. Natl Academy of Sciences, 23;98(22):12566-71, Oct 2001. Abstract