Advisor(s)

Phyllis R. Strauss (1943-)

Contributor(s)

Joanne Chan, Wendy A. Smith

Date of Award

2009

Date Accepted

8-2009

Degree Grantor

Northeastern University

Degree Level

M.S.

Degree Name

Master of Science

Department or Academic Unit

College of Arts and Sciences. Department of Biology.

Keywords

Biology, Molecular, DNA polymerase beta, Methyl methanesulfonate

Subject Categories

DNA polymerases, Logperch, DNA repair

Disciplines

Developmental Biology | Molecular Biology

Abstract

Base excision repair (BER) is the most common DNA repair mode in nature. It is very important for repairing alkylation damage, oxidative damage and AP sites produced by either environmental or endogenous agents. The BER pathway and many of its key components have been very well characterized by in vitro reconstituted biochemical assays using mammalian recombinant proteins. DNA polymerase beta (Pol ß) is the major DNA polymerase in this pathway. The bifunctional enzyme, which has both 5'-dRP lyase and DNA synthesis activities, is indispensable for mammalian development, but not cell survival. However, the early lethality in PolB-/- mice prevents a further investigation of the mechanism underlying death shortly after parturition. In this study, I investigated the role of Pol ß in early development using a vertebrate model, zebrafish. I first cloned the zebrafish polb gene and characterized its expression pattern at both mRNA and protein levels throughout zebrafish embryogenesis. Functional studies using both knockdown and overexpression were performed thereafter. Zebrafish polb encodes a very conserved protein, sharing ∼,80% sequence identity with its human counterpart. Only one gene copy for polb is found in zebrafish genome, and one transcript is present in embryos and adults. Despite the fact that Pol ß mRNA is expressed at early developmental stages I examined, Pol ß protein is not detectable until after mid-gastrulation stage. The protein level then increases as development progresses. Although morphogenesis in zebrafish embryos was not affected by either reduction or overexpression of Pol ß in very early embryos, Pol ß knockdown embryos showed a hypersensitivity phenotype to the alkylation agent, methyl methanesulfonate (MMS). Ectopic expression of eGFP-tagged Pol ß protein in the early knockdown embryos can partially restore MMS-resistance. These results imply that the Pol ß-dependent BER pathway is crucial for combating alkylation DNA damage, maintaining genome integrity, and promoting organismic survival. The protein that precedes Pol ß in the BER pathway is AP endonuclease (AP endo), which cleaves an abasic site to generate the 3'-hydroxyl group required by the polymerase. A reduction of Pol ß protein levels was found in AP endo hypomorphic zebrafish embryos generated by MO knockdown. In contrast, there was no change in AP endo protein levels after downregulating Pol ß. This finding suggests a potential regulatory function of AP endo on its downstream partner Pol ß. In short, this is the first study on the zebrafish polb gene. Expression studies and functional characterization during early development confirm its definitive role in DNA repair. However, knockdown zebrafish embryos develop normally during embryogenesis, implying that zebrafish Pol ß does not play an essential role in early development.

Document Type

Master's Thesis

Rights Holder

Xiaojie Yang


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