9/1/2023 0 Comments A human okazaki fragment![]() Association of FEN1 with the DNA under the different reaction conditions (Lower Panel). An asterisk on the right indicates the position of an aberrant ligation generated as a result of incomplete gap filling synthesis (see Fig. The ligated product appears as a doublet that is most likely due to secondary structure in the ligated oligonucleotide under the gel electrophoresis conditions. The positions of the labeled primer (Primer), the gap filled product (Fully Extended), longer products generated by strand displacement synthesis (Strand Displacing) and the ligated product (Ligated) are indicated on the left. After separation by denaturing gel electrophoresis, labeled DNA reaction products were detected and quantitated by phosphorimager analysis (Upper panel). In reactions with labeled FEN1, individual proteins (−) and dNTPs (−, no dNTPs −T, no TTP) were omitted as indicated. Reconstitution assays were carried out as described in Fig. Representative gel of at least three independent experiments with error bars showing standard deviation and with significance calculated and described as indicated in Materials and Methods. S3, labeled Pol δ was not retained by the streptavidin beads in the absence of the biotinylated DNA.The binding of labeled Pol δ to the DNA is expressed as the ratio of Pol δ retained on the beads to the DNA recovered on the beads. Association of Pol δ with the DNA under the different reaction conditions. After separation by denaturing gel electrophoresis, labeled DNA reaction products were detected and quantitated by phosphorimager analysis. In reactions with labeled Pol δ, individual proteins (−) and dNTPs (−, no dNTPs −T, no TTP) were omitted as indicated. In the fully reconstituted reaction, the labeled DNA substrate (5’P, 1 pmol) with a 30-nucleotide gap and a 5-nucleotide 5’-flap was pre-incubated with 4.5 pmol LacR and 1 pmol RPA prior to incubation with the indicated proteins (1 pmol of each except for 2.5 pmol PCNA trimer) as shown. Schematic diagram of reconstitution assay. Thus, dynamic PCNA complexes coordinate Okazaki fragment synthesis and processing with PCNA and LigI forming a terminal structure of two linked protein rings encircling the ligated DNA.ĭNA flap cleavage DNA ligation DNA replication DNA synthesis DNA-protein complex.Ĭopyright © 2020 Elsevier Ltd. Following ligation, PCNA and LigI remain on the DNA, indicating that Pol δ and FEN1 dissociate during 5' end processing and that LigI engages PCNA at the DNA nick generated by FEN1 and Pol δ. Both LigI and FEN1 associate with PCNA-Pol δ during gap-filling synthesis, suggesting that gap-filling synthesis is carried out by a complex of PCNA, Pol δ, FEN1 and LigI. Here we describe a novel interaction between Pol δ and LigI that is critical for Okazaki fragment joining in vitro. Although PCNA interacts with the enzymes DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (LigI) that complete Okazaki fragment processing and joining, it is not known how the activities of these enzymes are coordinated. After synthesis of an RNA-DNA oligonucleotide by DNA polymerase α 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). More than a million Okazaki fragments are synthesized, processed and joined during replication of the human genome.
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