The replisome is a complex molecular machine that uses cellular DNA as a template to produce an identical copy. Because of the orientation of the two DNA strands relative to the working direction of the polymerase, the leading strand is synthesized continuously, but the lagging strand is synthesized in segments termed Oka-zaki fragments. The bacterial replisome was long assumed to contain two polymerases, one each for the lagging strands. Recent studies, however, have provided evidence for three polymerases at Escherichia coli replication forks. Why three polymerases would be required remained unclear. Georgescu et al. prepared replisomes containing either two or three polymerases and used single-molecule total internal fluorescence microscopy to monitor DNA synthesis. Tripoly-merase replisomes were more processive than dipolymerase replisomes, synthesizing products that were nearly twice as long. Differences in DNA synthesis were greater on the lagging than the leading strand, and examination of DNA products showed that the dipolymerase replisome left single-strand gaps, whereas the tripolymerase replisome filled in the lagging strand much more efficiently.
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