Brendler T, Austin S
ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA.
EMBO J. 1999 Apr 15;18(8):2304-10. doi: 10.1093/emboj/18.8.2304.
The SeqA protein binds to the post-replicative forms of the origins of replication of the Escherichia coli chromosome (oriC) and the P1 plasmid (P1oriR) at hemimethylated GATC adenine methylation sites. It appears to regulate replication by preventing premature reinitiation. However, SeqA binding is not exclusive to replication origins: different fragments with hemimethylated GATC sites can bind SeqA in vitro when certain rules apply. Most notably, more than one such site must be present on a bound fragment. The protein appears to recognize individual hemimethylated sites, but must undergo an obligate cooperative interaction with a nearby bound protein for stable binding. SeqA contacts both DNA strands in a discrete patch at each hemimethylated GATC sequence. All four GATC bases are contacted and are essential for binding. Although the recognized sequence is symmetrical, the footprint on the methylated strand is always broader, suggesting that the bound protein is positioned asymmetrically with its orientation dictated by the position of the unique methyl group. Studies of alternative spacings and relative orientations of adjacent sites suggest that each site may be recognized by a symmetrical dimer with an induced asymmetry in one of the subunits similar to that seen with certain type II restriction endonucleases.
SeqA蛋白在半甲基化的GATC腺嘌呤甲基化位点与大肠杆菌染色体复制起点（oriC）和P1质粒（P1oriR）的复制后形式结合。它似乎通过防止过早重新起始来调节复制。然而，SeqA的结合并不局限于复制起点：当某些规则适用时，具有半甲基化GATC位点的不同片段在体外可以结合SeqA。最值得注意的是，一个结合片段上必须存在不止一个这样的位点。该蛋白似乎识别单个半甲基化位点，但必须与附近结合的蛋白进行强制性的协同相互作用才能稳定结合。SeqA在每个半甲基化GATC序列的一个离散区域内与两条DNA链接触。所有四个GATC碱基都被接触，并且对于结合至关重要。尽管识别的序列是对称的，但甲基化链上的足迹总是更宽，这表明结合的蛋白以不对称方式定位，其方向由独特甲基的位置决定。对相邻位点的替代间距和相对方向的研究表明，每个位点可能被一个对称二聚体识别，其中一个亚基中存在诱导的不对称性，类似于某些II型限制性内切酶的情况。
