Processing of lagging-strand intermediates in vitro by herpes simplex virus type 1 DNA polymerase.
Processing of lagging-strand intermediates in vitro by herpes simplex virus type 1 DNA polymerase.
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单纯疱疹病毒 1 型 DNA 聚合酶在体外加工滞后链中间体。
DOI：
10.1128/jvi.01875-09
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发表时间：
2010
影响因子：
5.4
通讯作者：
Parris,DeborahS
中科院分区：
文献类型：
--
作者：
Zhu,Yali;Wu,Zetang;Cardoso,MCristina;Parris,DeborahS
1型单纯疱疹病毒（HSV-1）的滞后链中间体的处理尚未在体外得到证实。在野生型或外切酶缺陷型（exo−）HSV-1 DNA聚合酶（pol）存在的情况下，检测了人皮瓣内切酶-1 （Fen-1）与模型滞后链中间体产生可连接产物的能力。引物/模板由一个小圆单链DNA模板与含有5 ‘ DNA襟翼或5 ’退火DNA或RNA序列的引物退火组成。野生型HSV-1 pol延长了缺口DNA引物/模板，但没有明显的链移位，尽管exo - HSV-1 pol观察到明显的链移位。然而，与野生型或exo - HSV-1 pol和Fen-1孵育含有5 ' flap的引物/模板，可以有效地产生缺口，这些缺口可以用DNA连接酶i密封。两种聚合酶都刺激Fen-1在含有DNA或rna的引物/模板上的缺口翻译活性，表明活性是协调的。Fen-1参与HSV-1 DNA合成的进一步证据表明，瞬时表达的绿色荧光蛋白与Fen-1融合的能力能够在感染细胞的病毒DNA复制区中积累，以及内源性Fen-1能够与HSV-1感染细胞中必需的病毒DNA复制蛋白共免疫沉淀。
The processing of lagging-strand intermediates has not been demonstratedin vitrofor herpes simplex virus type 1 (HSV-1). Human flap endonuclease-1 (Fen-1) was examined for its ability to produce ligatable products with model lagging-strand intermediates in the presence of the wild-type or exonuclease-deficient (exo−) HSV-1 DNA polymerase (pol). Primer/templates were composed of a minicircle single-stranded DNA template annealed to primers that contained 5′ DNA flaps or 5′ annealed DNA or RNA sequences. Gapped DNA primer/templates were extended but not significantly strand displaced by the wild-type HSV-1 pol, although significant strand displacement was observed with exo−HSV-1 pol. Nevertheless, the incubation of primer/templates containing 5′ flaps with either wild-type or exo−HSV-1 pol and Fen-1 led to the efficient production of nicks that could be sealed with DNA ligase I. Both polymerases stimulated the nick translation activity of Fen-1 on DNA- or RNA-containing primer/templates, indicating that the activities were coordinated. Further evidence for Fen-1 involvement in HSV-1 DNA synthesis is suggested by the ability of a transiently expressed green fluorescent protein fusion with Fen-1 to accumulate in viral DNA replication compartments in infected cells and by the ability of endogenous Fen-1 to coimmunoprecipitate with an essential viral DNA replication protein in HSV-1-infected cells.