Wang E H, Prives C
Department of Biological Sciences, Columbia University, New York, New York 10027.
J Biol Chem. 1991 Jul 5;266(19):12668-75.
We have characterized the biochemical activities of purified polyoma (Py) large T antigen (T Ag) that was capable of mediating the replication of a plasmid containing the Py origin (ori(+) DNA) in mouse cell extracts. We report here that like the T Ag encoded by simian virus 40 (SV40), Py T Ag has DNA helicase and double-stranded DNA fragment unwinding activities. Py T Ag displaced DNA fragments greater than 1,600 nucleotides which were annealed to complementary sequences in single-stranded M13 by translocating in the 3' to 5' direction. Both helicase and double-stranded DNA fragment unwinding reactions were completely dependent upon NTP hydrolysis, displaying a strong preference for ATP and dATP. At low T Ag concentrations, significantly more Py ori(+) DNA fragment was unwound compared with a fragment lacking the replication origin. However, at higher ratios of Py T Ag to DNA, equivalent to those used in replication reactions, unwinding of both ori-containing and -lacking fragments was equally efficient. This is in contrast to SV40 T Ag which exhibited a more stringent requirement for SV40 origin sequences under similar conditions. Furthermore, some of the nucleotides that supported the helicase and unwinding activities of Py T Ag were different from those for the same SV40 T Ag reactions. We have also observed that in contrast to the very poor replication of linear SV40 ori(+) DNA by SV40 T Ag in human cell extracts, linear Py ori(+) DNA was replicated efficiently in mouse cell extracts by Py T Ag. However, despite the fact that linear Py ori(+), SV40 ori(+), and ori(-) DNA fragments could be unwound with comparable efficiency by Py T Ag, only fragments containing the Py replication origin were replicated in vitro. These results suggest that the initiation of DNA synthesis at the Py origin of replication requires features in addition to unwinding of the template.
我们已对纯化的多瘤病毒（Py）大T抗原（T Ag）的生化活性进行了表征，该抗原能够在小鼠细胞提取物中介导含有Py复制起点（ori(+) DNA）的质粒的复制。我们在此报告，与猿猴病毒40（SV40）编码的T Ag一样，Py T Ag具有DNA解旋酶和双链DNA片段解旋活性。Py T Ag通过在3'至5'方向上移位，置换了大于1600个核苷酸的DNA片段，这些片段与单链M13中的互补序列退火。解旋酶和双链DNA片段解旋反应都完全依赖于NTP水解，对ATP和dATP表现出强烈偏好。在低T Ag浓度下，与缺乏复制起点的片段相比，Py ori(+) DNA片段的解旋明显更多。然而，在与复制反应中使用的比例相当的更高Py T Ag与DNA比例下，含ori和不含ori的片段的解旋效率相同。这与SV40 T Ag形成对比，在类似条件下，SV40 T Ag对SV40起源序列有更严格的要求。此外，支持Py T Ag解旋酶和解旋活性的一些核苷酸与相同SV40 T Ag反应的核苷酸不同。我们还观察到，与SV40 T Ag在人细胞提取物中对线性SV40 ori(+) DNA的复制非常差相反，线性Py ori(+) DNA在小鼠细胞提取物中被Py T Ag有效复制。然而，尽管线性Py ori(+)、SV40 ori(+)和ori(-) DNA片段可以被Py T Ag以相当的效率解旋，但只有含有Py复制起点的片段在体外被复制。这些结果表明，在Py复制起点处的DNA合成起始除了模板解旋外还需要其他特征。
