EFFICIENT RESOLUTION OF REPLICATED POXVIRUS TELOMERES TO NATIVE HAIRPIN STRUCTURES REQUIRES 2 INVERTED SYMMETRICAL COPIES OF A CORE TARGET DNA-SEQUENCE
EFFICIENT RESOLUTION OF REPLICATED POXVIRUS TELOMERES TO NATIVE HAIRPIN STRUCTURES REQUIRES 2 INVERTED SYMMETRICAL COPIES OF A CORE TARGET DNA-SEQUENCE
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DOI：
10.1128/jvi.61.6.1957-1963.1987
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发表时间：
1987-06-01
影响因子：
5.4
通讯作者：
MCFADDEN, G
中科院分区：
文献类型：
--
作者：
DELANGE, AM;MCFADDEN, G
麻痘病毒Shope纤维瘤病毒(SFV)线性双链DNA基因组的末端发夹序列已在酿酒酵母和重组缺陷大肠杆菌中克隆，作为回文插入环状质粒载体。这种序列配置相当于在体内复制痘病毒时检测到的作为端粒复制中间产物的反向重复结构。以前，已经证明，当包含这个回文插入的环状质粒被转染到SFV感染的细胞中时，有效的复制和分辨产生了具有真正的病毒发夹末端的线性微小染色体(A.M.DeLange，M.Ready，D.SCraba，C.Upton，和G.McFadden，J.Virol。56：249-259,1986)。为了定位有效分辨所需的最小靶DNA序列，在反向重复序列的两端构建了一系列交错的单向缺失。对不同克隆的分辨效率的分析表明，最大限度地分辨微小染色体需要高达240个以对称轴为中心的碱基对。为了研究富含AT的中轴序列在SFV中的作用，在SFV中包括8个非回文的BP，利用了对称轴上唯一的AflII位点。构建了从该AflII位点延伸的双向缺失，并将合成的寡核苷酸插入其中一个缺失衍生物中，并在体内进行了测试。这些质粒分解成带有发夹末端的线性微染色体的效率使我们能够将最小的靶DNA序列定义为长度在58到76bp之间的相同DNA序列的两个倒置拷贝。非回文核苷酸，在解析后，构成了天然痘病毒端粒特有的螺旋外残基，不是解析所必需的。SFV核心靶标序列与正痘病毒痘苗病毒的类似区域非常相似，这与痘病毒端粒解析的保守机制一致。
The terminal hairpin sequences of the linear double-stranded DNA genome of the leporipoxvirus Shope fibroma virus (SFV) has been cloned in Saccharomyces cerevisiae and in recombination-deficient Escherichia coli as a palindromic insert within circular plasmid vectors. This sequence configuration is equivalent to the inverted repeat structure detected as a telomeric replicative intermediate during poxvirus replication in vivo. Previously, it has been shown that when circular plasmids containing this palindromic insert were transfected into SFV-infected cells, efficient replication and resolution generated linear minichromosomes with bona fide viral hairpin termini (A.M. DeLange, M. Ready, D. Scraba, C. Upton, and G. McFadden, J. Virol. 59:249-259, 1986). To localize the minimal target DNA sequence required for efficient resolution, a series of staggered unidirectional deletions were constructed at both ends of the inverted repeat. Analyses of the resolution efficiencies of the various clones indicate that up to 240 base pairs (bp) centered at the symmetry axis were required for maximal resolution to minichromosomes. To investigate the role of the AT-rich central axis sequences, which in SFV include 8 nonpalindromic bp, a unique AflII site at the symmetry axis was exploited. Bidirectional deletions extending from this AflII site and insertions of synthetic oligonucleotides into one of the deletion derivatives were constructed and tested in vivo. The efficiency with which these plasmids resolved to linear minichromosomes with hairpin termini has enabled us to define the minimal target DNA sequence as two inverted copies of an identical DNA sequence between 58 and 76 bp in length. The nonpalindromic nucleotides, which, after resolution, constitute the extrahelical residues characteristic of native poxviral telomeres, were not required for resolution. The close resemblance of the SFV core target sequence to the analogous region from the orthopoxvirus vaccinia virus is consistent with a conserved mechanism for poxviral telomere resolution.