Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin.
Sequence-specific interaction with the viral AL1 protein identifies a geminivirus DNA replication origin.
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DOI：
10.1105/tpc.4.7.799
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发表时间：
1992-07
期刊：
影响因子：
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通讯作者：
S. Lazarowitz;WU ai'LeejuC.;Stephen G. Rogerqb;-J.;Elmerb
中科院分区：
文献类型：
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作者：
S. Lazarowitz;WU ai'LeejuC.;Stephen G. Rogerqb;-J.;Elmerb
番茄黄金花叶病毒(TGMV)和南瓜曲叶病毒(SqLCV)等双体双生病毒具有两个单链环状基因组DNA，A和B组分，被认为是从双链环状DNA中间体复制而来的。虽然已经推测病毒复制的起始序列位于两个基因组成分中都存在的高度保守的200核苷酸公共区(CR)，并且病毒编码的AL1蛋白与这些序列相互作用而影响复制，但没有证据表明这是事实。我们已经研究了这些问题，展示了这种蛋白质-DNA相互作用的选择性和序列特异性。TGMV和SqLCV DNA之间的简单组分切换和叶盘复制分析表明，TGMV和SqLCV的A组分促进自身复制，而它们的同源B组分促进自身复制，但都不复制非同源B组分。此外，利用体内功能复制实验，我们发现克隆的病毒CR序列作为复制起点，并在AL1存在的情况下指导非病毒序列的复制，合成了环状单链和双链DNA。最后，通过创建嵌合的病毒CRS和病毒CR的特定亚片段，我们证明了AL1蛋白对复制起始点的序列特异性识别，从而将起始点定位到CR1近端的大约90个核苷酸片段，其中包括保守的双生病毒茎环结构和大约60个核苷酸的5‘上游序列。通过缺失分析，我们进一步证明了保守的茎环结构是复制所必需的。这些研究确定了CR内复制的功能性病毒来源，表明复制需要AL1蛋白对该来源的序列特异性识别。
The bipartite geminiviruses such as tomato golden mosaic virus (TGMV) and squash leaf curl virus (SqLCV) have two single-stranded circular genomic DNAs, the A and B components, thought to be replicated from double-stranded circular DNA intermediates. Although it has been presumed that the origin sequences for viral replication are located in the highly conserved 200-nucleotide common region (CR) present in both genomic components and that the viral-encoded AL1 protein interacts with these sequences to effect replication, there has been no evidence that this is in fact so. We have investigated these questions, demonstrating selectivity and sequence specificity in this protein-DNA interaction. Simple component switching between the DNAs of TGMV and SqLCV and analysis of replication in leaf discs showed that whereas the A components of both TGMV and SqLCV promote their own replication and that of their cognate B component, neither replicates the noncognate B component. Furthermore, using an in vivo functional replication assay, we found that cloned viral CR sequences function as a replication origin and direct the replication of nonviral sequences in the presence of AL1, with both circular single-stranded and double-stranded DNA being synthesized. Finally, by the creation of chimeric viral CRs and specific subfragments of the viral CR, we demonstrated sequence-specific recognition of the replication origin by the AL1 protein, thereby localizing the origin to an approximately 90-nucleotide segment in the AL1 proximal side of the CR that includes the conserved geminiviral stem-loop structure and approximately 60 nucleotides of 5' upstream sequence. By deletional analysis, we further demonstrated that the conserved stem-loop structure is essential for replication. These studies identify the functional viral origin of replication within the CR, demonstrating that sequence-specific recognition of this origin by the AL1 protein is required for replication.