DIFFERENCES IN THE MECHANISM OF STIMULATION OF T7 DNA-POLYMERASE BY 2BINDING MODES OF ESCHERICHIA-COLI SINGLE-STRANDED DNA-BINDING PROTEIN

Escherichia coli single-stranded DNA-binding protein (Eco SSB) has bee n shown previously to display several DNA binding modes depending on t he ionic conditions. To determine what effect these various binding mo des have on DNA replication, we have studied DNA synthesis by the T7 D NA polymerase under ionic conditions where Eco SSB interacts with eith er 72 or 91 nucleotides of M13 DNA. These forms presumably correspond to the previously described (SSB)(56) land (SSB)(65) (Lohman and Ferra ri, 1994) that were determined using the binding of SSB to homopolymer s. Here we report the stimulation induced by (SSB)(91) to be 4-fold gr eater than that produced by (SSB)(72) under conditions where the templ ate is in large excess. Surprisingly, when the polymerase level is rai sed so that it is in molecular excess, (SSB)(91) no longer stimulates synthesis while (SSB)(72) affords a 4-fold stimulation, which is the s ame level of stimulation as when the template was mi excess. Both SSB forms increase the rate of DNA synthesis and were found to stimulate s ynthesis 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 fo r the template.