Cerritelli S, Springhorn S S, Lacks S A
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Proc Natl Acad Sci U S A. 1989 Dec;86(23):9223-7. doi: 10.1073/pnas.86.23.9223.
The two DNA-adenine methylases encoded by the Dpn II restriction gene cassette were purified, and their activities were compared on various DNA substrates. DpnA was able to methylate single-strand DNA and double-strand DNA, whereas DpnM methylated only double-strand DNA. Although both enzymes act at 5'-GATC-3' in DNA, DpnA can also methylate sequences altered in the guanine position, but at a lower rate. A deletion mutation in the dpnA gene was constructed and transferred to the chromosome. Transmission by way of the transformation pathway of methylated and unmethylated plasmids to dpnA mutant and wild-type recipients was examined. The mutant cells restricted unmethylated donor plasmid establishment much more strongly than did wild-type cells. In the wild type, the single strands of donor plasmid DNA that enter by the transformation pathway are apparently methylated by DpnA prior to conversion of the plasmid to a double-strand form, in which the plasmid would be susceptible to the Dpn II endonuclease. The biological function of DpnA may, therefore, be the enhancement of plasmid transfer to Dpn II-containing strains of Streptococcus pneumoniae.
对由Dpn II限制基因盒编码的两种DNA腺嘌呤甲基化酶进行了纯化，并在各种DNA底物上比较了它们的活性。DpnA能够甲基化单链DNA和双链DNA，而DpnM仅甲基化双链DNA。尽管这两种酶都作用于DNA中的5'-GATC-3'，但DpnA也能甲基化鸟嘌呤位置发生改变的序列，不过速率较低。构建了dpnA基因的缺失突变体并将其转移到染色体上。研究了通过甲基化和未甲基化质粒的转化途径向dpnA突变体和野生型受体的传递。与野生型细胞相比，突变细胞对未甲基化供体质粒的建立限制要强得多。在野生型中，通过转化途径进入的供体质粒DNA单链在质粒转化为双链形式之前显然被DpnA甲基化，而在双链形式下质粒会易受Dpn II核酸内切酶的作用。因此，DpnA的生物学功能可能是增强质粒向含有Dpn II的肺炎链球菌菌株的转移。
