期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：27
页码：E3476-E3484
DOI：10.1073/pnas.1510280112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceFunctional classification of DNA polymerases (DNAPs) usually divides them into replicative faithful replicases and error-prone enzymes devoted to DNA repair and DNA damage tolerance through translesion synthesis (TLS). When we analyzed the biochemical properties of phage Bam35 replicative DNAP, we found it to be a highly faithful DNAP that can couple strand displacement to processive DNA synthesis, suitable for rolling circle amplification of plasmidic DNA. Interestingly, it is also endowed with intrinsic TLS capacity opposite abasic sites and processive primer extension beyond the lesion. These features configure a versatile enzyme for accurate maintenance of viral genomic information over generations and, besides, to deal with DNA lesions, which suggest a possible application of Bam35 DNAP for the amplification of damaged or ancient DNA. DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very low-fidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage {Phi}29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.
关键词：protein-primed DNA polymerase ; Bam35 ; abasic sites ; translesion synthesis ; isothermal DNA amplification
