Mechanisms of DNA Double-Strand Break Repair in Mammalian Cells

Parker, Elizabeth Jane McFie (1991) Mechanisms of DNA Double-Strand Break Repair in Mammalian Cells. PhD thesis, University of Glasgow.

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The DNA double strand break (DSB) is the lesion believed to be responsible for the cytotoxic effects of ionising radiation. A number of cell lines hypersensitive to ionising radiations have been shown to have a defect in DSB repair. However, the irs series of mutants derived from V79 hamster cells are extremely radiosensitive but show no defect in DSB repair detectable by neutral elution. Previous studies using transfection of plasmid containing a DSB induced by restriction endonucleases into these cell lines suggested that one of the irs mutants was deficient in the fidelity of rejoin of such a DSB. The present study describes an assay for the capacity of nuclear extracts prepared from radiosensitive and wild type cells to rejoin an endonuclease induced DSB in vitro. Endonuclease treatment was used to linearise plasmid DNA producing DSB substrates with either 3' or 5' cohesive termini which were incubated with nuclear extracts from wild type or radiosensitive cell lines. The efficiency and fidelity of DSB rejoining was asessed by bacterial transformation and Southern blot hybridisation of reaction products. Nuclear extracts prepared from V79 and irs-1, a radiosensitive mutant derived from V79, efficiently catalyse the faithful religation of an endonuclease induced DSB as measured by increase in bacterial transformation of plasmid DNA. The level of DSB rejoin is dependent on the amount of nuclear extract added. In comparison, nuclear extracts prepared from another radiosensitive mutant of V79, irs-2, are unable to rejoin such a DSB to give molecules capable of transforming bacteria. The addition of V79 nuclear extract to irs-2 is shown to compensate for the irs-2 defect in production of a transforming molecule. Incubation of linear plasmid with nuclear extracts prepared from V79 or irs-1 gives rise to a molecule migrating faster than the linear form on electrophoresis and resistant to l-exonuclease treatment. This form is presumed to be closed circular plasmid DNA. It is not detected after reaction of linear plasmid with nuclear extract prepared from irs-2 cells. However irs-2 is not deficient in all pathways of DSB ligation, since nuclear extracts from this line catalyse the rejoin of linear substrate to high molecular weight concatemers as efficiently as extracts prepared from wild type or irs-1 cells. These linear concatemer forms are the products of a ligation reaction proceding with equivalent efficiency and fidelity in nuclear extracts from all cell types. Consistent with the capacity of nuclear extracts from irs-2 cells to catalyse concatemer formation is the finding that the protein concentration response, inducibility and fractionation characteristics of DNA ligases I and II are comparable in extracts from irs-2 and V79 wild type cells. Experiments designed to address the role of concatemers as substrates for the production of a circular molecule by recombination failed to show a difference between irs-2 and V79. The addition of specific purified protein activities to irs-2 extracts in attempts to restore wild type activity are also described. No compensation of the irs-2 defect was observed on addition of T4 ligase, gyrase or topoisomerase I activities.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Oncology
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78256
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Feb 2020 12:09
Last Modified: 28 Feb 2020 12:09

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