DNA Replication

RESEARCH HIGHLIGHTS DNA REPLICATION Keeping up with the leader DNA replication occurs at the helicase) does not pause during DNA As the leading strand T7 repli- replication fork, which forms when synthesis, as previously thought, but some does not pause during primer the lagging DNA is unwound by a helicase the lagging strand polymerase syn- synthesis, this cannot explain why strand into strands that are copied by two thesizes DNA faster than the leading the leading and lagging strands polymerases into a leading strand strand polymerase. are synthesized at the same rate. polymerase and a lagging strand. The leading The authors investigated the kinet- However, the authors found that T7 synthesizes strand is synthesized in a continuous ics of RNA primer synthesis, DNA DNA polymerase synthesizes DNA DNA faster manner, whereas synthesis of the unwinding and DNA synthesis by the from a template mimicking the than the lagging strand requires a primase bacteriophage T7 replication complex unwound lagging strand 30% faster that makes RNA primers that are (the T7 replisome), which consists of than the T7 replisome, suggesting leading strand extended by the DNA polymerase the T7 gp4 (a protein that contains that the leading strand polymerase polymerase. to form Okazaki fragments — short a helicase and a primase domain) is slowed down by its association DNA fragments that are processed to and the T7 DNA polymerase, using with T7 gp4. This allows time for produce a continuous DNA strand. synthetic replication fork substrates. the lagging strand to attach to a Despite this complexity, the lagging Single molecular fluorescence new primer and initiate synthesis of strand is synthesized at the same rate resonance energy transfer (FRET) — another Okazaki fragment, and still as the leading strand, but how is this energy transfer between two fluoro- keep up with leading strand DNA achieved? Pandey et al. now reveal phores, which in this case are in close synthesis. that the leading strand polymerase proximity before the DNA strands This study suggests that primer (which works closely with the separate — shows a FRET decrease synthesis does not pause DNA as the fluorophores move apart upon synthesis, and that primers are made DNA unwinding and DNA synthesis, concomitant with DNA synthesis, by the same rate in both the presence kept close to the replication complex and absence of a priming sequence by the lagging strand priming loop that allows primer synthesis. This sug- and extended by the lagging strand gests that the T7 replisome does not polymerase faster than the T7 repli- pause during lagging strand primer some can copy the leading strand. synthesis. Pausing is not necessary Katharine H. Wrighton because the nascent lagging strand template loops out between the heli- ORIGINAL RESEARCH PAPER Pandey, M. et al. case and primase domains of T7 gp4 Coordinating DNA replication by means of priming loop and differential synthesis rate. so that it keeps the RNA primers near Nature 18 Nov 2009 (doi:10.1038/nature08611) to the T7 replisome. NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 11 | JANUARY 2010.

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