A PROTEIN THAT BINDS TO THE P1 ORIGIN CORE AND THE ORIC 13MER REGION IN A METHYLATION-SPECIFIC FASHION IS THE PRODUCT OF THE HOST SEQA GENE
A PROTEIN THAT BINDS TO THE P1 ORIGIN CORE AND THE ORIC 13MER REGION IN A METHYLATION-SPECIFIC FASHION IS THE PRODUCT OF THE HOST SEQA GENE
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DOI：
10.1002/j.1460-2075.1995.tb00080.x
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发表时间：
1995-08-15
期刊：
影响因子：
11.4
通讯作者：
AUSTIN, S
中科院分区：
文献类型：
--
作者：
BRENDLER, T;ABELES, A;AUSTIN, S
P1质粒复制起点P1 oriR由七聚体重复序列内的四个GATC腺嘌呤甲基化位点的甲基化控制。这两个起源对甲基化的反应相当;通过识别半甲基化DNA进行阴性对照我们已经分离了一种宿主蛋白，该蛋白仅当其分离自擅长腺嘌呤甲基化的菌株时才识别P1起始区，基本上纯化的22 kDa蛋白质也以甲基化特异性方式结合oriC的13聚体区域，它被证明是seqA基因的产物，该基因通过螯合作用在oriC的阴性控制中起作用，我们得出结论，SeqA蛋白在螯合中的作用是通过直接DNA结合来识别P1 oriR和oriC的甲基化状态，使用合成底物，我们表明SeqA仅与这些起源的半甲基化形式结合，这些形式是甲基化熟练菌株中复制的直接产物。我们还表明，该蛋白质可以识别多个GATC位点的序列，而不管周围的序列，起源特异性的基础主要是持久性的半甲基化形式，在天然DNA制剂相对于控制过度。
The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats, A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function, We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation, The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation-specific fashion, It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration, We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding, Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins, forms that are the immediate products of replication in a methylation-proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence, The basis for origin specificity is primarily the persistence of hemimethylated forms that are over-represented in the natural DNA preparations relative to controls.