Mechanismen der nicht homologen Endverknüpfung in Zellen der höheren Eukaryonten: Funktionen der DNA Ligase III und Substypen von Histone H1

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There is evidence that non homologous end joining (NHEJ) is central to the removal of DNA DSBs in higher eukaryotes. Our results suggest the operation of two components of NHEJ, a DNA-PK dependent component that operates with fast kinetics (D-NHEJ), and a DNA-PK-independent component (basic or B-NHEJ) that operates with slow kinetics and utilizes as yet unidentified factors. When D-NHEJ is inhibited, B-NHEJ rather than homology directed repair (HDR), takes over to remove DSBs from the genome. There is ample evidence that DNA ligase IV is involved in D-NHEJ. The central hypothesis of this proposal is that DNA ligase III is involved in B-NHEJ. The objective is to confirm this preliminary observation and to elucidate the role of DNA ligase III in B-NHEJ. In additiion, guided by very recent exciting observations we propose to investigate the roles of H1 family histones in NHEJ. We will: 1. Utilize an in vitro plasmid assay to characterize the function of DNA ligase III in B-NHEJ and will compare to that of DNA ligase IV in D-NHEJ. 2. Utilize RNA interference (RNAi) to knock down expression of DNA ligase III and study its manifestations on DSB rejoining and cell radiosensitvivty to killing. 3. Investigate the role of the histone H1 subtypes, H1.2 an H1X, in NHEJ. The knowledge to be acquired as part of these studies will advance our understanding of the mechanismus of DNA DASB repair and probably also of genomic stability.