DNA primase acts as a molecular brake in DNA replication.
A hallmark feature of DNA replication is the coordination between the continuous polymerization of nucleotides on the leading strand and the discontinuous synthesis of DNA on the lagging strand. This synchronization requires a precisely timed series of enzymatic steps that control the synthesis of an RNA primer, the recycling of the lagging-strand DNA polymerase, and the production of an Okazaki fragment. Primases synthesize RNA primers at a rate that is orders of magnitude lower than the rate of DNA synthesis by the DNA polymerases at the fork. Furthermore, the recycling of the lagging-strand DNA polymerase from a finished Okazaki fragment to a new primer is inherently slower than the rate of nucleotide polymerization. Different models have been put forward to explain how these slow enzymatic steps can take place at the lagging strand without losing coordination with the continuous and fast leading-strand synthesis. Nonetheless, a clear picture remains elusive. Here we use single-molecule techniques to study the kinetics of a multiprotein replication complex from bacteriophage T7 and to characterize the effect of primase activity on fork progression. We observe the synthesis of primers on the lagging strand to cause transient pausing of the highly processive leading-strand synthesis. In the presence of both leading- and lagging-strand synthesis, we observe the formation and release of a replication loop on the lagging strand. Before loop formation, the primase acts as a molecular brake and transiently halts progression of the replication fork. This observation suggests a mechanism that prevents leading-strand synthesis from outpacing lagging-strand synthesis during the slow enzymatic steps on the lagging strand.
Lee
Jong-Bong
JB
Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, 250 Longwood Avenue, Boston, Massachusetts 02115, USA.
Hite
Richard K
RK
Hamdan
Samir M
SM
Xie
X Sunney
XS
Richardson
Charles C
CC
van Oijen
Antoine M
AM
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics*; D019915:DNA Primase / Q000378:metabolism*; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008957:Models, Genetic; D013698:Templates, Genetic
D004279:DNA, Viral; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase