5′ → 3′ molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates
5′ → 3′ molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates
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DOI：
10.1021/bi0005761
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发表时间：
2000-10-03
期刊：
影响因子：
2.9
通讯作者：
Borowiec, JA
中科院分区：
文献类型：
--
作者：
Iftode, C;Borowiec, JA
人类复制蛋白A（hRPA）以前被认为是有效地结合48 bp的猿猴病毒40（SV40）的“假起源”（PO）底物，模拟的DNA结构中发现的SV40 T抗原起点（ori）复合物。为了了解hRPA在复制起始过程中的作用，我们研究了hRPA相互作用的PO序列和结构要求。结合和解旋被认为是最有效的，当两条链的中央8 nt的单链DNA（ssDNA）气泡区域包含一个聚嘧啶结构，这些活动成比例地减少时，气泡区域被替换为嘌呤道上的一条或两条链。检查的两个双链侧翼的重要性表明，早期基因侧包含的DNA结构特征位于一个双链转从气泡的突变显着影响hRPA的亲和力的基板。当存在于ori的背景下时，该序列的突变被认为对体外SV40 DNA复制和ori的变性具有显著影响，表明起始活性可以通过改变hRPA结合亲和力的cia作用元件来调节。使用含有8nt嘧啶或嘌呤臂的叉和突出端底物表明，hRPA与DNA的结合涉及特定的分子极性，其中初始hRPA结合发生在ssDNA底物的5'侧，然后在3'方向上延伸以产生稳定结合的hRPA。这些数据有影响的真核生物DNA复制的启动机制，以及对网站的新生链合成内的起源。
Human replication protein A (hRPA) was previously seen to efficiently bind a 48 bp simian virus 40 (SV40) "pseudo-origin" (PO) substrate that mimics a DNA structure found within the SV40 T antigen-origin (ori) complex. To understand the role of hRPA during the initiation of replication, we examined the PO sequence and structure requirements for hRPA interaction. Binding and unwinding were found to be most efficient when both strands of the central 8 nt single-stranded DNA (ssDNA) bubble region contained a polypyrimidine structure, with these activities proportionately reduced when the bubble region was replaced with a purine tract on one or both strands. Examination of the importance of the two duplex flanks indicates that the early gene side contains a DNA structural feature located one duplex turn from the bubble whose mutation significantly affects the affinity of hRPA for the substrate. When present in the context of ori, mutation of this sequence was seen to have significant effects on SV40 DNA replication in vitro and on the denaturation of ori, indicating that origin activity can be modulated by cia-acting elements which alter the hRPA binding affinity. Use of fork and overhang substrates containing 8 nt pyrimidine or purine arms demonstrates that hRPA binding to DNA involves a particular molecular polarity in which initial hRPA binding occurs on the 5' side of a ssDNA substrate, and then extends in the 3' direction to create a stably bound hRPA. These data have implications on the mechanism of the initiation of eukaryotic DNA replication as well as on the sites of nascent strand synthesis within the origin.