Differences in the mechanism of stimulation of T7 DNA polymerase by two binding modes of Escherichia coli single-stranded DNA-binding protein.

Abstract:

Escherichia coli single-stranded DNA-binding protein (Eco SSB) has been shown previously to display several DNA binding modes depending on the ionic conditions. To determine what effect these various binding modes have on DNA replication, we have studied DNA synthesis by the T7 DNA polymerase under ionic conditions where Eco SSB interacts with either 72 or 91 nucleotides of M13 DNA. These forms presumably correspond to the previously described (SSB)56 and (SSB)65 (Lohman and Ferrari, 1994) that were determined using the binding of SSB to homopolymers. Here we report the stimulation induced by (SSB)91 to be 4-fold greater than that produced by (SSB)72 under conditions where the template is in large excess. Surprisingly, when the polymerase level is raised so that it is in molecular excess, (SSB)91 no longer stimulates synthesis while (SSB)72 affords a 4-fold stimulation, which is the same level of stimulation as when the template was in excess. Both SSB forms increase the rate of DNA synthesis and were found to stimulate synthesis by relieving template secondary structures. However, (SSB)72 specifically increases strand displacement synthesis, while (SSB)91 stimulates synthesis by increasing the affinity of the polymerase for the template.

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