Enzymatic Mechanisms of DNA Synthesis

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Mammalian chromosomal DNA is synthesized at replication forks in which one strand is made as short segments, called Okazaki fragments, that are later joined. We are defining the mechanism of the joining reaction by reconstitution with purified mammalian proteins, and using genetic tools from S. cerevisiae, which employs a similar replication system. Okazaki fragments are initiated by a short segment of RNA and then elongated to about 100 nucleotides. By current models, polymerization from an upstream fragment displaces the RNA region of the downstream fragment into a flap. The flap is thought to be removed by Dna2 nuclease/helicase (Dna2p) and flap endonuclease (FEN1), prior to ligation. However, some evidence suggests that the flap is removed without Dna2p. We will use reconstitution to determine whether Dna2p improves the efficiency of the reactions leading to joining. We will also further define the mechanism of Dna2p to establish whether its 5' flap entry, helicase tracking and cleavage pattern indicate that it works in sequence with FEN1. FEN1 cleaves at the base of the flap to create the ligation substrate. We showed that it recognizes the 5' end of the flap and then moves to the base. In vivo, the flap can equilibrate by branch migration to a variety of intermediates, but only one is a FEN1 substrate. Quench flow kinetic analyses will determine whether FEN1 waits for the correct configuration or induces its formation. FEN1 has a flexible loop structure thought to be involved in tracking to the cleavage site. Analysis of mutants should reveal the role of this structure. A proposed model for expansion of repeat sequences in somatic cells implicates intermediate substrate structures formed during the flap removal. We recently partially reconstituted this type of expansion, concluding that a competition between reactions catalyzed by DNA ligase I and FEN1 determines expansion probability. To verify our conclusions, expansion mechanisms will be probed in vivo in S. cerevisiae using a plasmid in which reporter gene expression is disrupted by expansion. Cellular expression of DNA ligase I will be varied to determine the effects of competition between ligation and FEN1 cleavage. Mammalian long patch base excision repair uses the same proteins as Okazaki fragment processing. It also includes AP endonuclease (APE1) which we found to be a coordinating and stimulatory factor. APE1 stimulates both FEN1 and DNA ligase I, and promotes movement of the substrate through the pathway. Binding and kinetic analyses will be employed to determine the mechanisms of APE1 directed stimulation.