Tomka M A, Catalano C E
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
J Biol Chem. 1993 Feb 15;268(5):3056-65.
Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of the viral genome into a preformed capsid. The overexpression of bacteriophage lambda-terminase in Escherichia coli has been previously reported (Chow, S., Daub, E., and Murialdo, H. (1987) Gene (Amst.) 60, 277-289), and we present here a purification scheme for the isolation of milligram quantities of protein which is homogenous ( > 97%) as determined by SDS-polyacrylamide gel electrophoresis. lambda-Terminase is composed of the gene products of Nu1 and A. Using N-terminal amino acid sequence analysis of the purified protein, we have determined a subunit stoichiometry of 2 gpNu1 polypeptides/gpA molecule in terminase holoenzyme. The circular dichroism spectrum for the purified holoenzyme has been obtained and is consistent with a protein complex composed primarily of alpha-helical structure. The endonucleolytic activity of the enzyme (the TER reaction) has been optimized with respect to pH, salt, and polyamine concentrations. Divalent metal ion is strictly required for the reaction and may be satisfied by either magnesium or manganese, but not by any of the other metals examined. E. coli integration host factor in amounts stoichiometric with the DNA substrate stimulates the TER reaction, but only when the enzyme is present in limiting amounts. Increasing the enzyme/DNA ratio attenuates the observed stimulation by integration host factor. A kinetic analysis of the TER reaction suggests that the assembly of multiple terminase promoters is required for efficient cleavage of viral DNA and that this reaction appears to be stoichiometric, rather than catalytic under the reaction conditions utilized. The implications of these results with respect to the packaging of viral DNA by terminase enzymes are discussed.
末端酶是复杂双链DNA病毒中常见的酶，是将病毒基因组包装到预先形成的衣壳中所必需的。此前已有报道在大肠杆菌中噬菌体λ末端酶的过表达（Chow，S.，Daub，E.，和Murialdo，H.（1987年）《基因》（阿姆斯特丹）60，277 - 289），我们在此展示一种纯化方案，用于分离毫克量的蛋白质，通过SDS - 聚丙烯酰胺凝胶电泳测定其纯度大于97%。λ末端酶由Nu1和A的基因产物组成。通过对纯化蛋白质进行N端氨基酸序列分析，我们确定了末端酶全酶中2个gpNu1多肽/gpA分子的亚基化学计量比。已获得纯化全酶的圆二色光谱，其与主要由α - 螺旋结构组成的蛋白质复合物一致。该酶的内切核酸酶活性（TER反应）已针对pH、盐和多胺浓度进行了优化。反应严格需要二价金属离子，镁或锰均可满足要求，但其他所检测的金属均不能满足。与DNA底物化学计量的大肠杆菌整合宿主因子可刺激TER反应，但仅当酶的量有限时才会如此。增加酶/DNA比例会减弱整合宿主因子所观察到的刺激作用。TER反应的动力学分析表明，多个末端酶启动子的组装是有效切割病毒DNA所必需的，并且在所用反应条件下该反应似乎是化学计量的，而非催化性的。讨论了这些结果对于末端酶包装病毒DNA的意义。
