Khan S A, Hayes S J, Wright E T, Watson R H, Serwer P
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Virology. 1995 Aug 1;211(1):329-31. doi: 10.1006/viro.1995.1411.
Both rate zonal centrifugation and gel electrophoresis have revealed that the mature double-stranded DNA of bacteriophage T5 has single-stranded breaks (nicks) at specific sites. Neither of these procedures has previously revealed site-specific nicks in the double-stranded DNA of other bacteriophages, including T7. In the present study, denaturing gel electrophoresis, followed by specific DNA detection, reveals that a small fraction of mature T7 DNA molecules, like most T5 DNA molecules, has site-specific nicks. The procedure of specific detection is to probe with an oligonucleotide specific for one of the ends of T7 DNA. If position 0.0 is the left genetic end and position 100.0 is the right genetic end of T7 DNA, the nicks on the 5' left-oriented strand are at 11.3, 12.4, 65.7, 79.2, and 86.0; the nicks on the 5' right-oriented strand are at 23.3 and 26.5 (+/- 0.5). The positions of the three rightmost nicks are indistinguishable from those of double-stranded breaks that produce previously demonstrated shorter than mature length DNAs packaged in vivo. We propose that the T7 nicks are produced by premature activity of the T7 terminase during DNA packaging.
速率区带离心法和凝胶电泳均已表明，噬菌体T5的成熟双链DNA在特定位点存在单链断裂（切口）。此前，这两种方法均未揭示包括T7在内的其他噬菌体双链DNA中的位点特异性切口。在本研究中，变性凝胶电泳结合特异性DNA检测表明，一小部分成熟的T7 DNA分子，如同大多数T5 DNA分子一样，具有位点特异性切口。特异性检测的方法是用针对T7 DNA一端的寡核苷酸进行探针检测。如果T7 DNA的0.0位置是左遗传末端，100.0位置是右遗传末端，那么5' 向左链上的切口位于11.
