Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase

被引:37
作者
Hwang, Y
Feiss, M
机构
[1] Department of Microbiology, University of Iowa, Iowa City
关键词
virus assembly; virus DNA packaging; genome encapsidation; DNA translocation; concatemer processing;
D O I
10.1006/jmbi.1996.0480
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimulated endonuclease activity of terminase is located in gpA, the large terminase subunit. There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490. Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase. In the present work, mutations causing the gpA changes K(497)A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo. Purified K(497)A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in cos cleavage are sufficient to explain the lethality of the K(497)A and K497D defects. In in vitro packaging studies using mature (cleaved) phage DNA, the K(497)A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 mu M ATP, indicating that the K497D change does not affect DNA translocation. In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase. (C) 1996 Academic Press Limited
引用
收藏
页码:524 / 535
页数:12
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