Data are smoothed to 5 bp.The DNA replication fork. The log 2 ratio of long to short fragments is plotted. Equal numbers of reads were selected from the top (long), and the bottom three (short) length quartiles of sequenced fragments. d, Okazaki fragments at nucleosome-depleted regions are disproportionately likely to be long. Data are aligned to TSSs such that the direction of transcription is from left to right: only Okazaki fragments synthesized in the same direction as transcription are analysed here equivalent analyses for fragments synthesized in the opposite direction can be found in Supplementary Fig. c, The distribution of Okazaki fragment termini correlates with nucleosome occupancy around TSSs. Unless otherwise indicated, all analyses use unsmoothed data normalized to the maximum signal in the analysed range, and area aligned such that Okazaki fragment (OF) synthesis by Pol δ proceeds from left to right. cerevisiae nucleosome dyad locations is shown. The distribution of termini from mononucleosome-sized Okazaki fragments around the top 50% consensus S. b, Okazaki fragment termini are enriched around nucleosome dyads. The inability to label fragments after ligase treatment confirms that labelled Okazaki fragments are flanked by ligatable nicks.Ī, Schematic indicating the mature Okazaki fragment termini sequenced in this study. Purified DNA was treated (lanes 2 and 4) or mock-treated (lanes 1 and 3) with the indicated ligase, and then labelled as in Fig. d, Okazaki fragments are bordered by ligatable nicks. Okazaki fragments appear upon release of the culture into S phase (lanes 3 and 4). Cells were arrested in G1 using α-factor, during which time CDC9 transcription was inhibited by the addition of doxycycline, and degradation of the protein stimulated by activation of the degron system using galactose and 37 ☌. c, Okazaki fragments accumulate during S phase. The chromatin digestion patterns in lanes 4 and 5 indicate that CDC9 repression does not alter global chromatin structure. 1a nucleosomes were prepared from wild-type (lane 3) or repressible CDC9 strains (lanes 4 and 5) by in vivo Micrococcal Nuclease (MNase) digestion. Okazaki fragments (lane 2) were labelled as in Fig. b, The size of labelled Okazaki fragments mirrors the nucleosome repeat. Purified genomic DNA was labelled using exonuclease-deficient Klenow fragment and α- 32P dCTP and separated in a denaturing agarose gel. Cells carrying a doxycycline-repressible allele of the CDC9 gene were treated with doxycycline (Dox) for the indicated time. Our studies represent the first high-resolution analysis-to our knowledge-of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.Ī, Transcriptional repression of DNA ligase I ( CDC9) results in the accumulation of nicked DNA. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments.
0 Comments
Leave a Reply. |