DNA polymerase II (ε) of Saccharomyces cerevisiae dissociates from the DNA template by sensing single-stranded DNA
DNA polymerase II (ε) of Saccharomyces cerevisiae dissociates from the DNA template by sensing single-stranded DNA
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DOI：
10.1074/jbc.273.33.21332
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发表时间：
1998-08-14
影响因子：
4.8
通讯作者：
Sugino, A
中科院分区：
文献类型：
--
作者：
Maki, S;Hashimoto, K;Sugino, A
从酵母细胞中纯化了两种形式的酿酒酵母DNA聚合酶II（PCR），Pol II* 和Pol II。Pol II* 是含有256-kDa催化多肽的四亚基复合物，而Pol II仅由源自Pol II* 的256-kDa多肽的N-末端一半的145-kDa多肽组成。我们表明，Pol II* 和Pol II是不可区分的DNA链延伸的持续合成能力和速率。Pol Ⅱ * 和Pol Ⅱ与DNA模板复合物的平衡解离常数表明，这两种复合物的稳定性几乎相同。然而，当使用单链DNA作为解离的聚合酶的陷阱测量Pol II* 和Pol II从DNA模板的解离速率时，Pol II* 的解离比Pol II快75倍。此外，随着单链DNA浓度的增加，Pol II* 从DNA模板解离的速率变得更快。这些结果表明，单链DNA积极促进Pol II* 从DNA模板的快速解离。单链DNA也促进Pol II从DNA模板的解离，尽管速率慢得多。这些结果表明，用于感测单链DNA的位点位于催化亚基的145-kDa N-末端部分内，并且通过该位点感测单链DNA的效率由催化亚基和/或其他亚基的C-末端一半正调制。
Two forms of DNA polymerase II (epsilon) of Saccharomyces cerevisiae, Pol II* and Pol II, were purified to near homogeneity from yeast cells. Pol II* is a four-subunit complex containing a 256-kDa catalytic polypeptide, whereas Pol II consists solely of a 145-kDa polypeptide derived from the N-terminal half of the 256-kDa polypeptide of Pol II*. We show that Pol II* and Pol II are indistinguishable with respect to the processivity and rate of DNA-chain elongation. The equilibrium dissociation constants of the complexes of Pol II* and Pol II with the DNA template showed that the stability of these complexes is almost the same. However, when the rates of dissociation of the Pol II* and Pol II from the DNA template were measured using single-stranded DNA as a trap for the dissociated polymerase, Pol II* dissociated 75-fold faster than Pol II. Furthermore, the rate of dissociation of Pol II* from the DNA template became faster as the concentration of the single-stranded DNA was increased. These results indicate that the rapid dissociation of Pol II* from the DNA template is actively promoted by single-stranded DNA, The dissociation of Pol II from the DNA template was also shown to be promoted by single-stranded DNA, although at a much slower rate. These results suggest that the site for sensing single stranded DNA resides within the 145-kDa N-terminal portion of the catalytic subunit and that the efficiency for sensing single-stranded DNA by this site is positively modulated by either the C-terminal half of the catalytic subunit and/or the other subunits.