The T4 replication machinery is functionally equivalent to that used by humans and employs eleven phage-encoded proteins, including gene 43 DNA polymerase, gene 61 primase, gene 41 helicase, gene 59 helicase loading protein, genes 44/62 and 45 polymerase accessory proteins, gene 32 single-stranded DNA binding protein, T4 RNase H, and T4 DNA ligase. Our previous electron microscopy revealed a single complex of proteins present on DNA replicated by the bacteriophage T4 proteins (obtained in collaboration with Dr. Jack Griffith at University of North Carolina Chapel Hill). This complex was found at the interface between replicated and non-replicated DNA, the so-call replication fork, and it is thought to contain the leading and lagging strand proteins. Surprisingly the looped out single-stranded DNA predicted in the trombone was coated with gene 32 binding protein and folded into a compact structure on a majority of the replicating molecules. This structure may help to explain how this trombone loop is efficiently duplicated by a single complex of replication proteins. We have now probed the composition of this replication complex using nanoscale DNA biopointers to show the location of biotin-tagged replication proteins. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the polymerases responsible for leading and lagging strand replication are held together in a single complex. Under fixation conditions in which the protein bound lagging strand template is teased apart, occasional molecules show two nascent lagging strand fragments, each being elongated by a biotin-tagged polymerase, suggesting that individual polymerase molecules may disassociate from the rapidly advancing replication fork to ensure complete replication of lagging strand fragments. This may explain why inhibitors of lagging strand synthesis do not greatly affect the rate of leading strand synthesis. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules but on a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase-loading protein is present on molecules with extensive replication, suggesting that this protein remains on the fork after loading the helicase or that forks collapse during replication and require 59 protein to restart them.
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