Biomedical Sciences ETDs

Author

Leyma De Haro

Publication Date

5-1-2009

Abstract

Metnase is a recently evolved human protein with methylase (SET) and nuclease domains that is widely expressed, especially in proliferating tissues. Metnase promotes plasmid and viral DNA integration, and through an interaction with topoisomerase IIα (TopoIIα) it promotes chromosome decatenation. Metnase interacts with DNA ligase IV, promotes non-homologous end-joining (NHEJ), and repression causes mild hypersensitivity to ionizing radiation. TopoIIα has a proposed role in relaxing positive supercoils in front of replication forks. NHEJ factors have been implicated in the replication stress response. Here we show that Metnase promotes cell proliferation, but does not affect replication fork elongation as measured by cell cycle analysis, BrdU incorporation and DNA fiber analysis. Even though there is no elongation effect, Metnase confers resistance to three replication stress agents, hydroxyurea, UV light, and the topoisomerase I inhibitor, camptothecin. Metnase expression also increases the rate at which H2AX phosphorylation (a marker of stalled or collapsed replication forks) is resolved. There was no difference in formation of gamma-H2AX foci after exposure to these agents. Metnase coimmunoprecipitates (co-IP) with proliferating cell nuclear antigen (PCNA) and RAD9. Finally, we show that Metnase promotes TopoIIα-mediated relaxation of positively supercoiled DNA, similar to the torsional strain preceding replication forks. These results establish Metnase as an important component of the human replication stress response.

Keywords

Metnase, DNA replication stress, DNA damage, non-homologous end joining, hydroxyurea, replication fork

Sponsors

National Institutes of Health

Document Type

Dissertation

Language

English

Degree Name

Biomedical Sciences

Level of Degree

Doctoral

Department Name

Biomedical Sciences Graduate Program

First Advisor

Hromas, Robert

First Committee Member (Chair)

Osley, Mary Ann

Second Committee Member

Nickoloff, Jac

Third Committee Member

Hudson, Laurie

Fourth Committee Member

Chackerian, Bryce

Fifth Committee Member

Williamson, Elizabeth

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