Mammalian cells have a marked ability to repair double strand breaks in chromosomal DNA, but the pathways by which this is accomplished, and the detailed biochemical mechanisms, of the repair reactions are not known. A detailed description of these pathways would lead to a better understanding of the rearrangement of the genome during the development of the immune system and the creation of chromosomal abnormalities in tumorigenesis. Distinguishing the non-homologous from the homologous pathways also may be of practical benefit in accomplishing homologous replacement of defective genes by their wild type counterparts, or in the experimental disruption of genes for functional studies. This proposal is an extension to the biochemical level of previous studies which aimed to describe the fate of adenoviral DNA transmitted to the cell by infection or transfection. Left and right terminal fragments of adenoviral DNA with different restriction-cut ends, will be transfected into human A549 cells, virus recovered, and the junctions sequenced. The pattern of sequences will suggest possible mechanisms for the joining of ends with complementary, blunt, and non-complementary sequences. A recently developed crude nuclear extract will be optimized for end- joining linear plasmid DNA, in particular the joining of blunt to blunt and 3' to blunt ends, as these are efficiently repaired in mammalian cells, but are either inefficiently joined, or refractory to joining, by known ligases. The junctions will be characterized either directly from the products of the in vitro reaction, or after transformation into E. coli. Evidence will be sought for the formation of heteroduplex DNA at the site of joining. The proteins responsible for efficient end-joining will be purified from the crude nuclear extracts by column chromatography and reconstitution experiments to try to reproduce the joining events seen in the viral transfections and the crude extract reactions. Homologous recombinational repair of double strand breaks in plasmid DNA, will be studied using crude nuclear extracts followed by bacterial transformation. Different extraction and fractionation procedures are proposed to try to increase the efficiency of the reaction, so that detailed biochemical characterization can be performed. If this is successful, the physical structures formed in the reaction and the kinetics of their formation resolution, can be determined.
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