Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis.
In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.
Hamdan
Samir M
SM
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Loparo
Joseph J
JJ
Takahashi
Masateru
M
Richardson
Charles C
CC
van Oijen
Antoine M
AM
D017123:Bacteriophage T7 / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics; D004261:DNA Replication / Q000502:physiology*; D004279:DNA, Viral / Q000032:analysis / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000378:metabolism; D013997:Time Factors; D000099272:Replisomes
D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D009097:Multienzyme Complexes; D000099272:Replisomes