It is of utmost importance for every organism to maintain the integrity of its genome, both in germ line cells to ensure viable and genetically healthy offspring, as well as in somatic cells for the proper development and health of the adult organs and tissues. A very drastic type of DNA damages are double-strand breaks caused by ionizing radiation. Such breaks can cause loss of entire chromosome segments or, if improperly repaired, can lead to chromosome rearrangements. Such major damage to the genome is likely to lead to cell death or cell transformation. The repair of double-stranded DNA breaks requires a process called illegitimate recombination or non-homologous DNA end-joining (NHEJ). In humans and other mammalian species genetic defects have been identified that cause hypersensitivity to ionizing radiation. Some of these radiation sensitive organisms and cells display at the same time severe combined immunodeficiency (SCID). These cells cannot repair double-strand breaks and also cannot carry out the rearrangement of the immunoglobulin and T-cell receptor genes (VDJ recombination) during lymphoid development, a process that is similar to NHEJ. Recent studies showed that mutations causing this type of radiosensitivity are located in the genes encoding the three components of the DNA-dependent protein kinase (DNA-PKcs), the 470-kD catalytic subunit and the Ku70/80 heterodimer. DNA-PK is an enzyme that is activated by binding to double-stranded DNA ends and preferentially phosphorylates substrates bound to the same DNA molecule. How DNA-PK is involved in repair of double-strand breaks in DNA and V(D)J recombination is not known and represents a major gap in knowledge. To address detailed mechanistic questions about the role of DNA-PK in DNA repair, genetic studies must be complemented with biochemical approaches. A cell-free extract from fertilized Xenopus eggs has been described that efficiently and precisely joins non-homologous DNA ends. The joining reaction is inhibited both after removal of Ku protein and in the presence of the DNA-PK inhibitor wortmannin. This in vitro system is therefore ideally suited to study molecular and mechanistic aspects of DNA-PK function during DNA end joining. The present application proposes (1) to define the DNA end structures that are joined in Ku-dependent and Ku-independent reactions, (2) to fractionate the egg extract into DNA-PK and other components required for the DNA end joining reaction and to reconstitute the activity with the fractions, and (3) to examine the dynamics of the interaction of DNA-PK with the repair complex. The proposed experiments will address the hypothesis that Ku is required to align non-matching DNA ends for the joining reaction and that the phosphorylation of a component of the repair machinery by DNA-PK is an essential step during the DNA end joining reaction.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Chemical Pathology Study Section (CPA)
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Wolfe, Paul B
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Torrey Pines Institute for Molecular Studies
San Diego
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