Cue D, Feiss M
Department of Microbiology, University of Iowa, Iowa City 52242.
J Mol Biol. 1992 Nov 5;228(1):72-87. doi: 10.1016/0022-2836(92)90492-3.
Terminase, the DNA packaging enzyme of phage lambda, binds to lambda DNA at a site called cosB, and introduces staggered nicks at an adjacent site, cosN, to generate the cohesive ends of virion lambda DNA molecules. Terminase also is involved in separation of the cohesive ends and in binding the prohead, the empty protein shell into which lambda DNA is packaged. Terminase is a DNA-dependent ATPase, and both subunits, gpNu1 and gpA, have ATPase activity. cosB contains a series of gpNu1 binding sites, R3, R2 and R1; between R3 and R2 is a binding site, I1, for integration host factor (IHF), the Escherichia coli DNA bending protein. In this work, a series of mutations in Nu1 have been isolated as suppressors of cosB mutations. One of the Nu1 mutations is identical to the previously described Nu1ms1/ohm1 mutation predicted to cause the change L40F in the 181 amino acid-long gpNu1. Three other Nu1 missense mutations, the Nu1ms2 (L40I), ms3 (Q97K) and ms4 (A92G) mutations, have been isolated; the relative strengths of suppression of cosB mutations by the Nu1ms mutations are: ms1 > ms2 > ms3 > ms4. The Nu1 missense mutations all affect amino acid residues that lie outside of the putative helix-turn-helix DNA binding motif of gpNu1. The Nu1ms1 and Nu1ms2 mutations alter an amino acid residue (L40) that lies directly between two segments of gpNu1 proposed to be involved in ATP binding and hydrolysis; thus these mutations are likely to alter the gpNu1 ATP-binding site. The Nu1ms3 and Nu1ms4 mutations both affect amino acid residues in the central region of gpNu1 that is predicted to form a hydrophilic alpha-helix. To explain how the Nu1ms mutations suppress cosB defects, models involving alterations of the DNA binding and/or catalytic properties of terminase are considered. The results also indicate that terminase occupancy of a single gpNu1 binding site (R3) is necessary and sufficient for the efficient initiation of DNA packaging; the Nu1ms1, ms2 and ms3 mutations permit IHF-independent plaque formation by a phage lacking R2 and R1.
噬菌体λ的DNA包装酶——末端酶，在一个名为cosB的位点与λ DNA结合，并在相邻位点cosN处引入交错切口，以产生病毒粒子λ DNA分子的粘性末端。末端酶还参与粘性末端的分离以及与原头部（λ DNA被包装入的空蛋白壳）的结合。末端酶是一种依赖DNA的ATP酶，其两个亚基gpNu1和gpA都具有ATP酶活性。cosB包含一系列gpNu1结合位点，即R3、R2和R1；在R3和R2之间是整合宿主因子（IHF，一种大肠杆菌DNA弯曲蛋白）的结合位点I1。在这项研究中，已分离出一系列Nu1中的突变作为cosB突变的抑制子。其中一个Nu1突变与先前描述的预测会导致181个氨基酸长的gpNu1中L40F变化的Nu1ms1/ohm1突变相同。还分离出了其他三个Nu1错义突变，即Nu1ms2（L40I）、ms3（Q97K）和ms4（A92G）突变；Nu1ms突变对cosB突变的抑制相对强度为：ms1 > ms2 > ms3 > ms4。Nu1错义突变均影响位于gpNu1假定的螺旋-转角-螺旋DNA结合基序之外的氨基酸残基。Nu1ms1和Nu1ms2突变改变了一个氨基酸残基（L40），该残基直接位于gpNu1中两个被认为参与ATP结合和水解的片段之间；因此，这些突变可能会改变gpNu1的ATP结合位点。Nu1ms3和Nu1ms4突变均影响gpNu1中央区域中预计会形成亲水性α-螺旋的氨基酸残基。为了解释Nu1ms突变如何抑制cosB缺陷，考虑了涉及改变末端酶的DNA结合和/或催化特性的模型。结果还表明，末端酶占据单个gpNu1结合位点（R3）对于DNA包装的有效起始是必要且充分的；Nu1ms1、ms2和ms3突变允许缺乏R2和R1的噬菌体形成不依赖IHF的噬菌斑。
