Advisor(s)

Phyllis Strauss

Abstract

Oxidative damage to DNA is a byproduct of normal processes essential for life, and therefore subsequent repair is necessary to maintain cell vitality. Damage to DNA causes incorrect base pairing that can lead to nonfunctional proteins and consequently result in cell abnormalities, cancer or death. Base excision repair (BER) functions to repair non-bulky lesions, uracil, and sites lacking a nucleobase. The pathway can utilize one of two mechanisms known as short or long-patch repair in which there is single or multiple base insertion respectively. BER begins with the recognition and removal of the incorrect or damaged base via a DNA glycosylase. The resulting apurinic/apyrimidinic site (AP site) is cleaved with AP endonuclease. In short patch repair, a DNA polymerase mediates subsequent insertion of a single correct base and removal of the deoxyribose phosphate group (dRP). In long patch repair, a replicative DNA polymerase inserts 2-6 bases and the resulting displaced 3’ strand is cleaved by flap endonuclease 1 (FEN1). Finally, the DNA strand is sealed with DNA ligase. Several of these key enzymes are embryonic lethals in knockout mice. Although BER is well characterized in adult tissues and cells, its role during development is poorly understood. Since the pathway has never been investigated during embryogenesis, we characterized BER in zebrafish extracts from unfertilized eggs, embryos at different developmental stages and adults. Using a 45-mer double-stranded substrate with a U/G mispair at position 21, we showed that extracts from all stages are capable of performing BER. Before 3 dpf aphidicolin-sensitive polymerases perform most nucleotide insertion. After hatching at 3 dpf, an aphidicolin-resistant polymerase, probably DNA polymerase β, becomes the primary polymerase. Previously we showed that when zebrafish AP endonuclease protein (ZAP1) level is knocked down, embryos cease dividing after the initial phase of rapid proliferation and die without apoptosis shortly thereafter. Nevertheless, extracts from embryos in which ZAP1 has been largely depleted process substrate equally as well as extracts from control embryos. Since apex1 and apex2 are both strongly expressed in early embryos relative to adults, these data indicate that both may play important roles in DNA repair in early development. In brief, early stage embryos are fully capable of cleaving an AP site after substantial depletion of AP endonuclease and of inserting repair deoxynucleotides despite a lack of detectable DNA polymerase β protein. Therefore, the major differences in BER performed by early stage embryos and adults are the predominance of replicative polymerases and the presence of backup Mg²⁺-dependent endonuclease activity in early stage embryos. The switch to normal, adult BER occurs when embryos hatch from the chorionic membrane and encounter normal oxidative stress.

Date Accepted

Spring 5-2009

Publication Date

4-1-2009

Degree Grantor

Notheastern University

Disciplines

Life Sciences

Rights Holder

Sean M. Fortier

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Included in

Life Sciences Commons

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