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Nucleic Acids Research, 1985, Vol. 13, No. 7 2559-2568
© 1985

Articles

{alpha}-Putrescinylthymine and the sensitivity of bacteriophage {varphi}W-14 DNA to restriction endonucleases

Patricia Black Miller, W.W. Wakarchuk and R.A.J. Warren

Department of Microbiology, University of British Columbia, Vancouver, British Columbia V6T 1W5, Canada

Received December 10, 1984. Revised February 27, 1985. Accepted February 27, 1985.

The modified base {alpha}-putrescinylthymine (putT) in {varphi}W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving {varphi}w-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase.


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