Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2011 Aug 12;286(32):28488-97. doi: 10.1074/jbc.M111.252023. Epub 2011 Jun 23.
DNA polymerase and DNA helicase are essential components of DNA replication. The helicase unwinds duplex DNA to provide single-stranded templates for DNA synthesis by the DNA polymerase. In bacteriophage T7, movement of either the DNA helicase or the DNA polymerase alone terminates upon encountering a nick in duplex DNA. Using a minicircular DNA, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling circle DNA synthesis. A gap in the non-template strand cannot be bypassed. The efficiency of bypass synthesis depends on the DNA sequence downstream of the nick. A nick on the template strand cannot be bypassed. Addition of T7 single-stranded DNA-binding protein to the complex stimulates nick bypass 2-fold. We propose that the association of helicase with the polymerase prevents dissociation of the helicase upon encountering a nick, allowing the helicase to continue unwinding of the duplex downstream of the nick.
DNA 聚合酶和 DNA 解旋酶是 DNA 复制的必需组成部分。解旋酶解开双链 DNA，为 DNA 聚合酶提供单链模板进行 DNA 合成。在噬菌体 T7 中，DNA 解旋酶或 DNA 聚合酶的单独运动在遇到双链 DNA 中的缺口时都会终止。使用一个小型环状 DNA，我们表明，尽管在非模板链上的效率降低了 7%，但解旋酶-聚合酶复合物可以绕过缺口，继续进行滚环 DNA 合成。非模板链上的缺口不能被绕过。旁路合成的效率取决于缺口下游的 DNA 序列。模板链上的缺口不能被绕过。将 T7 单链 DNA 结合蛋白添加到复合物中可将缺口旁路合成的效率提高 2 倍。我们提出，解旋酶与聚合酶的结合防止了解旋酶在遇到缺口时的解离，从而使解旋酶能够继续解开缺口下游的双链 DNA。
