Molecular anatomy and regulation of a stable replisome eukaryotic DNA at a paused replication fork

Arturo Calzada, Ben Hodgson, Masato Kanemaki, Avelino Bueno, Karim Labib

    Research output: Contribution to journalArticle

    197 Citations (Scopus)

    Abstract

    Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases alpha and epsilon and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.

    Original languageEnglish
    Pages (from-to)1905-1919
    Number of pages15
    JournalGenes & Development
    Volume19
    Issue number16
    DOIs
    Publication statusPublished - 2005

    Keywords

    • Mrc1
    • DNA replication forks
    • Tof1
    • S-PHASE
    • SCHIZOSACCHAROMYCES-POMBE
    • recombination
    • checkpoint
    • RIBOSOMAL DNA
    • Csm3
    • MINICHROMOSOME MAINTENANCE PROTEIN
    • RNA-POLYMERASE-I
    • RIBONUCLEOTIDE REDUCTASE
    • BUDDING YEAST
    • HELICASE ACTIVITY
    • MITOTIC RECOMBINATION
    • SACCHAROMYCES-CEREVISIAE

    Cite this

    Calzada, Arturo ; Hodgson, Ben ; Kanemaki, Masato ; Bueno, Avelino ; Labib, Karim. / Molecular anatomy and regulation of a stable replisome eukaryotic DNA at a paused replication fork. In: Genes & Development. 2005 ; Vol. 19, No. 16. pp. 1905-1919.
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    abstract = "Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases alpha and epsilon and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.",
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    Molecular anatomy and regulation of a stable replisome eukaryotic DNA at a paused replication fork. / Calzada, Arturo; Hodgson, Ben; Kanemaki, Masato; Bueno, Avelino; Labib, Karim.

    In: Genes & Development, Vol. 19, No. 16, 2005, p. 1905-1919.

    Research output: Contribution to journalArticle

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    T1 - Molecular anatomy and regulation of a stable replisome eukaryotic DNA at a paused replication fork

    AU - Calzada, Arturo

    AU - Hodgson, Ben

    AU - Kanemaki, Masato

    AU - Bueno, Avelino

    AU - Labib, Karim

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    AB - Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases alpha and epsilon and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.

    KW - Mrc1

    KW - DNA replication forks

    KW - Tof1

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    KW - SCHIZOSACCHAROMYCES-POMBE

    KW - recombination

    KW - checkpoint

    KW - RIBOSOMAL DNA

    KW - Csm3

    KW - MINICHROMOSOME MAINTENANCE PROTEIN

    KW - RNA-POLYMERASE-I

    KW - RIBONUCLEOTIDE REDUCTASE

    KW - BUDDING YEAST

    KW - HELICASE ACTIVITY

    KW - MITOTIC RECOMBINATION

    KW - SACCHAROMYCES-CEREVISIAE

    U2 - 10.1101/gad.337205

    DO - 10.1101/gad.337205

    M3 - Article

    VL - 19

    SP - 1905

    EP - 1919

    JO - Genes & Development

    JF - Genes & Development

    SN - 0890-9369

    IS - 16

    ER -