Assessing Biological Function of DNA Damage Response Genes

Larry H. Thompson

Lawrence Livermore National Laboratory

Why This Project

To understand the relative importance of individual DNA repair and DNA-damage response pathways to the recovery of mammalian cells after exposure to low doses of ionizing radiation (IR). This understanding may lead to better ways of setting limits on human exposure to IR. In spite of the discovery of many mammalian DNA repair genes, our current knowledge of how many of these genes contribute to cellular recovery from IR exposure is quite limited.

Project Goals

1. Measure cellular responses at doses in the 5-100 cGy range, which generally cause changes too small to detect in normal, repair-proficient cells
2. Focus on DNA double-strand breaks (DSBs) and DNA oxidative base damage plus single-strand breaks (SSBs)
3. Clarify the coordination of repair by cell-cycle checkpoint genes
4. Determine the relative contribution of the homologous recombinational and base-excision repair (BER) pathways in preventing IR-induced chromosomal rearrangement and instability.

Experimental Approach

We are focusing on two types of DNA damages induced by IR, DNA, DSB and DNA oxidative base damage plus SSBs. These damages are removed by the pathways of homologous recombinational repair (HRR) and BER, respectively. DNA DSB (which are also removed by an end-joining pathway) are considered the biologically most critical lesion produced by radiation, resulting in mutation, chromosomal rearrangements, and cell-killing. By constructing knockout mutations in the genetically stable CHO hamster cell line, we can evaluate the relative contribution of repair genes and pathways that are likely to be the most important in protecting cells against low dose radiation. In particular, the pathways and genes we are emphasizing are homologous recombinational repair (RAD51C and RAD51D), (APE1), and cell-cycle checkpoint genes (RAD1 and RAD17). To overcome the generally low efficiency of gene targeting in mammalian cells, highly sensitive PCR-based screening procedures are being utilized to detect gene disruption events. Mutant cell lines will be characterized for low dose radiation responses by measuring a combination of chromosomal aberrations in the G2 phase, and in some cases, SSB repair by the single-cell comet assay. Our initial evidence suggests that the dose response for chromosomal aberrations in an HRR mutant (XRCC2) may be nonlinear, with proportionately fewer aberrations occurring at higher doses (50-100 cGy) versus lower doses (10-20 cGy). As genetic variants are identified by others for the genes we are studying, we will evaluate their possible dysfunction by comparing normal versus variant expression constructs for their ability to complement (correct) the repair deficiency of the respective knockout mutant cell lines.

Expected Outcomes

Understand relevance, if any, of common genetic variants of human DNA repair genes to radiation susceptibility.

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