The clamp loader replication factor C (RFC) is essential for polymerase switching and for restricting Pol α from rebinding to Okazaki fragments even in the absence of Pol δ [ Replacing Pol α in the replisome with Pol δ, a processive and highly faithful polymerase, is crucial for restricting unwanted Pol α action. One important role during Okazaki fragment synthesis is to limit the function of Pol α because of its low processivity and lack of proofreading capability. Key mediators have been identified for dynamic protein–protein interactions and protein post-translational modifications which coordinate the access of different OFM enzymes to replication sites. This process requires the enzyme activities of Primase, Pol α, Pol δ, FEN1, and Lig I, as well as the precise coordination of their sequential actions at Okazaki fragment sites. In both short-flap and long-flap OFM, if the Pol α errors are not removed by 5′ flap cleavage, they can be edited out by the exonuclease activity of FEN1.Įfficient and proper OFM is essential for preventing prolonged exposure of ssDNA gaps, which may be converted into the most lethal and mutagenic double-strand breaks (DSBs), on the lagging-strand template. The process that removes the long 5′ flap by the sequential actions of DNA2 and FEN1 (or EXO1) is called long-flap OFM. The short flap is then cleaved by FEN1 or EXO1, and the DNA nick is ligated by Lig I. However, RPA recruits DNA2, which cleaves the long 5′ flap in the middle and creates a short 5′ flap (less than 10 nt). The ssDNA-binding protein RPA binds to the long RNA–DNA flap, inhibiting FEN1 cleavage of the 5′ flap. On the other hand, if PIF1 helicase is recruited to the replisome, DNA strand displacement is enhanced, leading to the formation of a long 5′ flap. If a short RNA–DNA flap escapes cleavage by FEN1 or EXO1, continuous DNA strand displacement may result in a long 5′ flap of up to 400 nt in length. The created ligatable DNA nick is then ligated by Lig I. The displacement–cleavage cycle continues until the whole RNA–DNA primer is degraded, which also removes any Pol α errors (dark dots in the RNA–DNA primer). EXO1 also degrades a relatively small percentage of such short 5′ flaps and serves as a backup of FEN1. In most cases the 5′ flap is 2–10 nt in length (short flap) and is primarily cleaved by FEN1, creating a DNA nick for ligation. Pol δ-mediated Okazaki fragment extension encounters an RNA–DNA primer in a downstream Okazaki fragment and displaces the primer portion to form a 5′ flap. Multiple nuclease-driven pathways for proper RNA–DNA primer removal We discuss new discoveries that implicate OFM enzymes as targets to specifically kill human cancer cells via synthetic lethality. In addition, over the past few years increasing evidence has emerged to indicate that prolonged existence of DNA single-strand breaks (SSBs) is a major cause of cell death in human cancer cells, which are frequently deficient in one or more DNA damage response and repair proteins. In particular, we review the most recent studies on how cells transform unprocessed RNA–DNA primers to induce alternative pathways for OFM and survival. We then discuss various DNA intermediate structures caused by faulty OFM processes and the induction of DNA damage checkpoints, and corresponding consequences for cell fate. In this review we first summarize current advances in understanding the distinct roles of various 5′ nucleases in RNA–DNA primer removal and the molecular mechanisms by which various OFM enzymatic reactions are coordinated through dynamic post-translational modifications and protein–protein interactions. Deletion of genes encoding key enzymes, regulatory proteins, or protein-modifying enzymes of OFM result in unligated Okazaki fragments and incomplete DNA replication, thus leading to cell death. Efficient and faithful OFM is crucial for genome integrity and cell survival.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |