Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation

[摘要] More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase a holoenzyme, proliferating cell nuclear antigen (PCNA), a homotrimeric DNA sliding clamp and polymerase processivity factor, is loaded onto the primer-template junction by replication factor C (RFC). Although PCNA interacts with the enzymes DNA polymerase delta (Pol delta), flap endonuclease 1 (FEN1) and DNA ligase I (Ligl) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. Here we describe a novel interaction between Pol delta and Ligl that is critical for Okazaki fragment joining in vitro. Both Ligl and FEN1 associate with PCNA-Pol delta during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol delta, FEN1 and Ligl. Following ligation, PCNA and Ligl remain on the DNA, indicating that Pol delta and FEN1 dissociate during 5' end processing and that Ligl engages PCNA at the DNA nick generated by FEN1 and Pol delta. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and Ligl forming a terminal structure of two linked protein rings encircling the ligated DNA. (C) 2020 Elsevier Ltd. All rights reserved.

[发布日期] 2020-12-04

[关键词] DNA replication;DNA synthesis;DNA flap cleavage;DNA ligation;DNA-protein complex