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Fork Reversal and ssDNA Accumulation at Stalled Replication Forks Owing to Checkpoint Defects

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Science  26 Jul 2002:
Vol. 297, Issue 5581, pp. 599-602
DOI: 10.1126/science.1074023
  • Figure 1

    Representative RIs isolated from in vivo psoralen cross-linked chromatin. Electron micrographs of replicating bubbles from wild-type (A) or rad53 (Band C) cells (27). The transition point from dsDNA to ssDNA is indicated by arrows (B). Black arrowheads indicate the single-stranded arms of the hemi-replicated bubbles; white arrowheads indicate the replicated strands (C and D). Graphic representation of the data in table S2 for hydroxyurea (HU)-treated or untreated (no treatment, NT) wild-type andrad53 cells for the number of hours (h) shown (E and F).

  • Figure 2

    Representative RIs purified as in Fig. 1 and analyzed under denaturing conditions. Replicating molecules from wild-type (A) and rad53 cells (B toD). The density of single-stranded bubbles representing nucleosomes (10) is similar in parental and daughter strands (table S2). Asterisks indicate large gaps at the forks (B and C). In (B), a fork containing single-stranded regions in both arms is visualized (asterisk and diamond). Black arrowheads indicate the single-stranded arms of the hemi-replicated bubbles; white arrowheads indicate the replicated strands (C and D). (A to D) Replicated arms are organized in single-stranded bubbles reflecting nucleosome assembly [see drawing in (C)].

  • Figure 3

    RIs containing four-way junctions from HU-treated rad53 cells. In most of the Holliday junctions formed at the replication forks, the four single strands involved in the branch point are visualized [(A), (B), (C), (G)]. (A to F) Samples were prepared under nondenaturing conditions. Arrows indicate single-stranded regions [(C) and (D)]. (G) and (H) Samples were prepared under denaturing conditions. In (H), the fourth arm is organized in single-stranded bubbles characteristic of nucleosomal DNA.

  • Figure 4

    DNA primase mutants accumulate hemi-replicated intermediates. Representative replicating bubbles frompri1-M4 mutant cells (A and B) (27). The molecule in (A) is partially single-stranded. Arrows indicate the transition points from dsDNA to ssDNA. In (B), the white arrowhead indicates the replicated arm of a hemi-replicated bubble; the black arrowhead indicates the unreplicated strand. (C) Schematic representation of RIs in wild-type and rad53 cells treated with HU or not treated. In HU-treated wild-type cells, the accumulation of short single-stranded regions likely causes checkpoint activation. In HU-treatedrad53 cells, abnormal replication intermediates, likely caused by a defect in the DNA polymerase α-primase complex, are converted into the aberrant structures represented in the gray panel. O, replication origin.

  • Table 1

    Classes and size of replication intermediates in the presence of hydroxyurea (HU). Data related to Figs. 1 and 2. Total number of analyzed RIs is in parentheses. wt, wild type.

    Cells and treatmentBubbles (%)Y-shaped molecules + broken bubbles (%)RI (n)Size of replicating bubbles (bp)RI (n)
    wt + HU 0.5 hours71.328.7(230)3025  ±  1732(149)
    wt + HU 1 hours74.525.5 (141)4227  ±  1807(100)
    wt + HU 2 hours7624(104)7484  ±  4155(76)
    rad53 + HU 0.5 hours70.829.2 (181)2099  ±  1681(106)
    rad53 + HU 1 hours77.522.5 (165)2747  ±  1355(87)
    rad53 + HU 2 hours45.854.2 (144)3116  ±  1991(56)

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    Fork Reversal and ssDNA Accumulation at Stalled Replication Forks Owing to Checkpoint Defects
    José M. Sogo, Massimo Lopes, and Marco Foiani

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