STUDY OF THE STRUCTURE OF REPLICATIVE INTERMEDIATES OF HSV-1 DNA BY PULSED-FIELD GEL-ELECTROPHORESIS
STUDY OF THE STRUCTURE OF REPLICATIVE INTERMEDIATES OF HSV-1 DNA BY PULSED-FIELD GEL-ELECTROPHORESIS
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DOI：
10.1006/viro.1994.1206
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发表时间：
1994-05-01
期刊：
影响因子：
3.7
通讯作者：
TYRRELL, DLJ
中科院分区：
文献类型：
--
作者：
SEVERINI, A;MORGAN, AR;TYRRELL, DLJ
通过脉冲场凝胶电泳将 HSV-1 感染细胞的 DNA 分离成两条病毒特异性条带：一条以线性单体基因组 (152 kb) 的形式迁移，另一条保留在凝胶的起点。后一条带含有复制的 HSV-1 DNA，通过 [H-3] 胸苷脉冲标记测定。为了研究这种“凝胶起源”DNA 的结构，我们构建了带有独特 PacI 限制位点的 HSV-1 KOS 突变体 (HSV-1 PAC1DTK)。感染后后期（24-48 小时）凝胶来源的 PAC1DTK DNA 的部分消化表明脉冲场凝胶电泳上存在线性多联体。在每个串联体中，相邻单体基因组的长 (L) 区域存在两种可能的方向。此外，在 U-L 区域的两个可能方向上检测到了尺寸与病毒基因组左端片段相对应的短于单位大小的片段。在感染后早期（8-12 小时），用 PacI 消化仅释放微量线性片段，并且大多数凝胶来源 DNA 在脉冲场凝胶电泳中不会迁移。为了释放从凝胶起点迁移的线性片段，需要使用 EcoRI（一种限制性内切酶，将 HSV-1 KOS 基因组切割 12 次）进行多次切割。这些结果表明，HSV-1 DNA 的复制中间体连接在一个大型网络中，需要在包装之前将其拆开。该网络可能由通过频繁重组事件连接在一起的线性分子组成，也可能由除简单滚环之外的复制模式（例如θ复制）的产物组成。 (C) 1994 年学术出版社
DNA from HSV-1-infected cells was separated by pulsed-field gel electrophoresis into two virus-specific bands: one that migrated as the linear monomer genome (152 kb) and another that remained at the origin of the gel. The latter band contained the replicating HSV-1 DNA, as determined by pulse-labeling with [H-3]thymidine. To investigate the structure of this ''gel origin'' DNA, we constructed a HSV-1 KOS mutant bearing a unique PacI restriction site (HSV-1 PAC1DTK). Partial digestion of gel origin PAC1DTK DNA at late times postinfection (24-48 hr) demonstrated the presence of linear concatemers on pulsed-field gel electrophoresis. Within each concatemer, the long (L) regions of adjacent monomer genomes were found in the two possible orientations. In addition, shorter-than-unit-size fragments that corresponded in size to the left end fragments of the viral genome were detected with the U-L region in the two possible orientations. At early times postinfection (8-12 hr), digestion with PacI released only a trace of linear fragments, and most of the gel origin DNA did not migrate on pulsed-field gel electrophoresis. Multiple cuts with EcoRI (a restriction enzyme that cuts the HSV-1 KOS genome 12 times) were necessary to release linear fragments that migrated from the origin of the gel. These results indicate that replicative intermediates of HSV-1 DNA are linked in a large network that needs to be unraveled before packaging takes place. This network may be composed of linear molecules linked together by frequent recombination events or of products of a mode of replication other than simple rolling circle (e.g., theta replication). (C) 1994 Academic Press, Inc.