Coordinating DNA replication by means of priming loop and differential synthesis rate

Article

关键词: LAGGING-STRAND SYNTHESIS;    RING-SHAPED HELICASE;    ESCHERICHIA-COLI;    MINICIRCLE SUBSTRATE;    REPLISOME;    PRIMASE;    PROCESSIVITY;    DEFICIENT;    MECHANISM;    TEMPLATE;   

DOI  :  10.1038/nature08611

来源: SCIE

【 摘 要 】

Genomic DNA is replicated by two DNA polymerase molecules, one of which works in close association with the helicase to copy the leading-strand template in a continuous manner while the second copies the already unwound lagging-strand template in a discontinuous manner through the synthesis of Okazaki fragments(1,2). Considering that the lagging-strand polymerase has to recycle after the completion of every Okazaki fragment through the slowsteps of primer synthesis and hand-off to the polymerase(3-5), it is not understood how the two strands are synthesized with the same net rate(6-9). Here we show, using the T7 replication proteins(10,11), that RNA primers are made 'on the fly' during ongoing DNA synthesis and that the leading-strand T7 replisome does not pause during primer synthesis, contrary to previous reports(12,13). Instead, the leading-strand polymerase remains limited by the speed of the helicase(14); it therefore synthesizes DNA more slowly than the lagging-strand polymerase. We show that the primase-helicase T7 gp4 maintains contact with the priming sequence during ongoing DNA synthesis; the nascent lagging-strand template therefore organizes into a priming loop that keeps the primer in physical proximity to the replication complex. Our findings provide three synergistic mechanisms of coordination: first, primers are made concomitantly with DNA synthesis; second, the priming loop ensures efficient primer use and hand-off to the polymerase; and third, the lagging-strand polymerase copies DNA faster, which allows it to keep up with leading-strand DNA synthesis overall.

【 授权许可】

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