Effective means of repairing DNA damage are essential for the survival of every organism. In eukaryotes, repair takes place in the context of a large genome heterogeneously compacted into chromatin. In order to study the mechanism of DNA repair by homologous recombination in the context of chromatin, assays must be established which allow manipulation of the reaction components. I propose to develop a set of assays that reconstitute the steps of homologous recombination in Xenopus laevis egg extracts. The most well characterized interaction between checkpoint control and chromatin structure is the phosphorylation of histone H2AX by the kinase ATM. ATM and H2AX form coincident foci with other repair factors in vivo. To determine the mechanistic role of ATM in directing DNA repair I will determine the requirements for both ATM and histone H2AX in repair, examine the dependence of other repair factors on ATM and H2AX and elucidate the changes in chromatin structure following H2AX phosphorylation.
