Berjón-Otero Mónica, Villar Laurentino, de Vega Miguel, Salas Margarita, Redrejo-Rodríguez Modesto
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):E3476-84. doi: 10.1073/pnas.1510280112. Epub 2015 Jun 22.
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 Φ29 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.
负责基因组复制的DNA聚合酶(DNAPs)是高度保真的酶,但仍无法处理受损的DNA。相比之下,跨损伤合成(TLS)DNAPs适合复制修饰的模板碱基,尽管会产生保真度非常低的产物。在这里,我们报告了温和噬菌体Bam35 DNA聚合酶(B35DNAP)的生化特性,它属于B族DNAPs的蛋白质引发亚组,与噬菌体Φ29以及其他病毒和移动元件聚合酶一起。B35DNAP是一种高度保真的DNAP,能够将链置换与连续DNA合成相结合。这些特性使其能够以非常低的错误率对质粒DNA进行多次置换扩增。尽管具有保真度和校对活性,B35DNAP仍能够成功地进行无模板重排的无碱基位点TLS,并优先在无碱基位点对面插入一个A(A规则)。此外,连续合成所需的TPR2亚结构域的缺失会损害引物在无碱基位点后的延伸。综上所述,这些发现表明B35DNAP可能在体内进行忠实且连续的基因组复制,并在需要时对无碱基位点进行TLS。