Replication of M13mp7 single‐stranded DNA in vitro by the 9‐S DNA polymerase α from calf thymus

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Abstract
The replication of phage M13 single-stranded DNA by the 9S DNA polymerase .alpha. from calf thymus was studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation were investigated. Oligonucleotides comprising only 4 nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single-stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase .alpha. contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the 4 nucleoside triphosphates. Priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA.

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