MULTICOMPONENT ORIGIN OF CYTOMEGALOVIRUS LYTIC-PHASE DNA-REPLICATION
MULTICOMPONENT ORIGIN OF CYTOMEGALOVIRUS LYTIC-PHASE DNA-REPLICATION
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DOI：
10.1128/jvi.65.2.931-937.1991
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发表时间：
1991-02-01
影响因子：
5.4
通讯作者：
PUNTURIERI, SM
中科院分区：
文献类型：
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作者：
ANDERS, DG;PUNTURIERI, SM
巨细胞病毒(CMV)裂解期DNA复制既需要反式作用因子，如病毒编码的DNA聚合酶，也需要以前未定义的顺式作用元件，即起始发生的起始点。我们已经确定了CMV裂解期DNA复制的候选来源oriLyt，通过评估所克隆的限制性片段在感染提供所需反式作用因子的情况下，在导入人成纤维细胞后介导磷酸甲酸敏感DNA复制的能力。在最初的实验中，类猴CMV株Colburn EcoRI D片段引导DNA复制；该片段包含所有单链DNA结合蛋白基因(DBP)和约7kbp的上游序列。人CMV DBP上游的较大区域也参与了瞬时检测中的复制。随后的亚克隆和缺失分析确定了CMV科尔伯恩株具有足够的起源功能，在DPB上游的明显非编码区跨越约1300bp。该区域的核苷酸序列包括四个不同的结构域，包括：(1)9个碱基重复序列，(2)富含A+T的片段，(3)11个碱基直接重复序列，(4)47个碱基直接重复序列。至少这些结构域中的某些部分是原始功能所必需的。因此，就像EB病毒裂解相源的DNA复制一样，CMV oriLyt似乎在结构上是复杂的。
Cytomegalovirus (CMV) lytic-phase DNA replication requires both trans-acting factors, such as the virus-coded DNA polymerase, and a previously undefined cis-acting element, the origin, within which initiation occurs. We have located a candidate origin of CMV lytic-phase DNA replication, oriLyt, in both simian and human strains by assessing the ability of cloned restriction fragments to mediate phosphonoformic acid-sensitive DNA replication after transfection into human fibroblasts when required trans-acting factors were supplied by infection. In initial experiments the simian CMV-like strain Colburn EcoRI D fragment directed DNA replication; this fragment contains all of the single-stranded DNA-binding protein gene (dbp) and about 7 kbp of upstream sequence. A larger region upstream of human CMV dbp also mediated replication in transient assays. Subsequent subcloning and deletion analyses defined a CMV strain Colburn region sufficient for origin function, spanning about 1,300 bp in the apparently noncoding region upstream of dpb. The nucleotide sequence of this region revealed four distinct domains, containing (i) a 9-bp repeated sequence, (ii) an A + T-rich segment, (iii) an 11-bp direct repeat, and (iv) a 47-bp direct repeat. At least some part of each of these domains was required for origin function. Therefore, like the Epstein-Barr virus lytic-phase origin of DNA replication, CMV oriLyt appears to be structurally complex.