WILLIAMS Brandi (USA) (Fellow)
JACKSON Stephen Philip (Host)
Department of Zoology, Wellcome / CRC Institute, Cambridge, UK
In vitro dissection of the non-homologous DNA end-joining repair pathway

DNA double strand breaks (DSBs) are introduced in the genome as either a consequence of normal metabolic processes or upon exposure to genotoxic agents. In mammalian cells, these potentially lethal DSBs are repaired predominantely by the non-homologous DNA end-joining (NHEJ) pathway. Genetic studies have identified four essential components of the pathway that include the 38 kDa product of the XRCC4 gene and the subunits of the DNA-dependent protein kinase, DNA-PK. DNA-PK is a nuclear protein that contains a 460 kDa catalytic subunit (DNA-PKcs) and a regulatory DNA-binding subunit known as Ku which is, itself, a heterodimer consisting of 70 kDa (Ku70) and 80 kDa (Ku80) polypeptides. The Ku complex binds DNA at the site of DSBs in a sequence-independent fashion. Current models suggest Ku functions in NHEJ to recruit DNA-PKcs to DNA ends, align the DNA ends for ligation and protect these ends from exonuclease activity and/or undergoing recombination. The binding of DNA-PKcs to DNA-bound Ku activates the serine/threonine kinase activity of the catalytic subunit. One possibility is that DNA-PKcs participates in NHEJ by directly phosphorylating and activating repair proteins. Alternatively, DNA-PKcs may recruit other components of the repair machinery to the sites of DNA damage.

The NHEJ pathway has recently been reconstituted in vitro using cell-free extracts from human lymphoblastoid cells. In this system, the ligation of DNA ends with complementary 3’ overhangs occurs without nucleotide loss or addition and is dependent upon all the factors known to be necessary for both NHEJ and V(D)J recombination. The data also indicate additional unknown factors are required for NHEJ. The overall objectives of my project are to characterize the function of DNA-PK in the NHEJ pathway and to identify novel components of this pathway by exploiting the newly defined in vitro NHEJ assay. A combination of immunodepletion/reconstitution studies as well as biochemical fractionation and purification using the NHEJ assay as a readout for function will be employed to obtain these goals.