Department of Microbe-Plant Interactions, CBIB (Center for Biomolecular Interactions Bremen), University of Bremen, D-28334 Bremen, Germany.
RNA. 2019 Jan;25(1):105-120. doi: 10.1261/rna.068486.118. Epub 2018 Oct 25.
Branchpoints in RNA templates are highly mutagenic, but it is not known yet whether this also applies to branchpoints in DNA templates. Here, we report how nucleic acid polymerases replicate a 2',5'-branched DNA (bDNA) molecule. We constructed long-chained bDNA templates containing a branch guanosine and T7 promoters at both arms by splinted ligation. Quantitative real-time PCR analysis was used to investigate whether a branchpoint blocks DNA synthesis from the two arms in the same manner. We find that the blocking effect of a branchpoint is arm-specific. DNA synthesis from the 2'-arm is more than 20,000-fold decreased, whereas from the 3'-arm only 15-fold. Our sequence analysis of full-length nucleic acid generated by Taq DNA polymerase, Moloney murine leukemia virus reverse transcriptase, and T7 RNA polymerase from the 2'-arm of bDNA shows that the branched guanine has a dual coding potential and can base-pair with cytosine and guanine. We find that branchpoint templating is influenced by the type of the surrounding nucleic acid and is probably modulated by polymerase and RNase H active sites. We show that the branchpoint bypass by the polymerases from the 3'-arm of bDNA is predominantly error-free, indicating that bDNA is not as highly mutagenic as 2',5'-branched RNA.
RNA 模板中的分支点具有高度的突变性，但目前尚不清楚这是否也适用于 DNA 模板中的分支点。在这里，我们报告了核酸聚合酶如何复制 2'，5'-分支 DNA（bDNA）分子。我们通过拼接连接构建了含有分支鸟苷和 T7 启动子的长链 bDNA 模板。通过定量实时 PCR 分析研究了分支点是否以相同的方式阻止两条臂上的 DNA 合成。我们发现分支点的阻断作用具有臂特异性。来自 2'-臂的 DNA 合成减少了 20000 多倍，而来自 3'-臂的仅减少了 15 倍。我们使用 Taq DNA 聚合酶、莫洛尼鼠白血病病毒逆转录酶和 T7 RNA 聚合酶从 bDNA 的 2'-臂生成全长核酸的序列分析表明，分支的鸟嘌呤具有双重编码潜力，可以与胞嘧啶和鸟嘌呤形成碱基对。我们发现分支点模板受到周围核酸类型的影响，并且可能通过聚合酶和 RNase H 活性位点进行调节。我们表明，聚合酶从 bDNA 的 3'-臂绕过分支点主要是无错误的，这表明 bDNA 的突变性不如 2'，5'-分支 RNA 高。
