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Nucleic Acids Research, 1986, Vol. 14, No. 8 3343-3362
© 1986

Articles

Persistence of DNA synthesis arrest sites in the presence of T4 DNA polymerase and T4 gene 32, 44, 45 and 62 DNA polymerase accessory proteins

Marc F. Charette1, David T. Weaver2 and Melvin L. DePamphilis

Department of Biological Chemistry. Harvard Medical School Boston, MA 02115, USA

Received December 9, 1985. Revised March 21, 1986. Accepted March 21, 1986.

DMA synthesis by phage T4 DNA polymerase is arrested at specific sequences in single–stranded DNA templates. To determine whether or not T4 DNA polymerase accessory proteins 32, 44, 45 and 62 eliminated recognition of these arrest sites, unique priner–templates were constructed in which DNA synthesis began at a DNA primer located at different distances from palindromic and nonpalindromic arrest sites. Nucleotide positions that caused polymerase to pause or leave the template were identified by sequence analysis of 5'–end labeled nascent DNA chains. Stable hairpin structures at palindromic sequences were confirmed by acetylation of single–stranded sequences with bromoacetaldehyde. Our results confirmed that these T4 DHA polymerase accessory proteins stimulated T4 DNA polymerase activity and processivity on natural as well as homopolymer primer-templates. However, they did not alter recognition of DNA synthesis arrest sites by T4 DNA polymerase. Extensive DHA synthesis resulted from an increased rate of translocation and/or processivity to the same extent over all DNA sequences.

1Present addresses: Creative Biomolecules, 35 South Street, Hopkinton, MA 01748

2Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge. MA 02142 USA


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