西亚试剂：Coordinating DNA replication by means of priming loop and d

发布时间：2025-06-23

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

Manjula Pandey1, Salman Syed2, Ilker Donmez1, Gayatri Patel1, Taekjip Ha2,3 & Smita S. Patel1

1 Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
2 Howard Hughes Medical Institute, Urbana, Illinois 61801, USA
3 Department of Physics and the Center for the Physics of Living Cells, University of Illinois, Urbana-Champaign, Illinois 61801, USA

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 fragments1, 2. Considering that the lagging-strand polymerase has to recycle after the completion of every Okazaki fragment through the slow steps of primer synthesis and hand-off to the polymerase3, 4, 5, it is not understood how the two strands are synthesized with the same net rate6, 7, 8, 9. Here we show, using the T7 replication proteins10, 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 reports12, 13. Instead, the leading-strand polymerase remains limited by the speed of the helicase14; 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.

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