High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin
High-mobility group 1/2 proteins are essential for initiating rolling-circle-type DNA replication at a parvovirus hairpin origin
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DOI：
10.1128/jvi.72.11.8477-8484.1998
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发表时间：
1998-11-01
影响因子：
5.4
通讯作者：
Tattersall, P
中科院分区：
文献类型：
--
作者：
Cotmore, SF;Tattersall, P
滚环复制由复制子编码的核酸内切酶启动，该核酸内切酶将单链切口引入特定的起始序列，在切口处共价连接到DNA的5'端，并提供3'羟基以引发单向的前导链合成。细小病毒例如小鼠微小病毒（MVM），已经采用这种机制，通过使用发夹端粒来扩增它们的线性单链基因组，所述发夹端粒顺序地展开和重折叠以使复制叉沿着基因组来回沿着，产生连续的多聚体DNA链。然后，病毒起始蛋白NS 1通过在发夹序列内存在的特定起点处产生切口并重新启动合成，从该连续体中切除单个基因组。使用体外试验研究右手（5 '）MVM发夹内的ATP依赖性起始，我们已经表征了HeLa细胞因子，该因子是允许NS 1在该起始处产生切口所绝对需要的。与细小病毒起始因子（PIF）不同，在左侧（3 '）病毒来源激活NS 1内切核酸酶活性的细胞复合物，激活右侧发夹的宿主因子在高盐中从磷酸纤维素洗脱，具有约25 kDa的分子量，并且似乎优先结合结构化DNA，提示它可能是高迁移率族1/2（HMG 1/2）蛋白家族的成员。证实了这一预测表明，纯化的小牛胸腺HMG 1和重组人HMG 1或鼠HMG 2可以分别取代HeLa因子，激活NS 1核酸内切酶的起源特异性切口反应。
Rolling-circle replication is initiated by a replicon-encoded endonuclease which introduces a single-strand nick into specific origin sequences, becoming covalently attached to the 5' end of the DNA at the nick and providing a 3' hydroxyl to prime unidirectional, leading-strand synthesis. Parvoviruses. such as minute virus of mice (MVM), have adapted this mechanism to amplify their linear single-stranded genomes by using hairpin telomeres which sequentially unfold and refold to shuttle the replication fork back and forth along the genome, creating a continuous, multimeric DNA strand. The viral initiator protein, NS1, then excises individual genomes from this continuum by nicking and reinitiating synthesis at specific origins present within the hairpin sequences. Using in vitro assays tea study ATP-dependent initiation within the right-hand (5') MVM hairpin, we have characterized a HeLa cell factor which is absolutely required to allow NS1 to nick this origin. Unlike parvovirus initiation factor (PIF), the cellular complex which activates NS1 endonuclease activity at the left-hand (3') viral origin, the host factor which activates the right-hand hairpin elutes from phosphocellulose in high salt, has a molecular mass of around 25 kDa, and appears to bind preferentially to structured DNA, suggesting that it might be a member of the high-mobility group 1/2 (HMG1/2) protein family. This prediction was confirmed by showing that purified calf thymus HMG1 and recombinant human HMG1 or murine HMG2 could each substitute for the HeLa factor, activating the NS1 endonuclease in an origin-specific nicking reaction.