ACTIVE-SITE OF THE REPLICATION PROTEIN OF THE ROLLING CIRCLE PLASMID PC194

被引:69
作者
NOIROTGROS, MF
BIDNENKO, V
EHRLICH, SD
机构
[1] Lab. de Genet. Microbienne, Inst. Natl. de la Rech. Agronomique
关键词
CATALYTIC CENTER; DNA REPLICATION; DNASE; EXONUCLEASE; TYPE I TOPOISOMERASE;
D O I
10.1002/j.1460-2075.1994.tb06761.x
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
Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from bl vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endo-nucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate, The former may be involved in the formation of the covalent DNA - protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.
引用
收藏
页码:4412 / 4420
页数:9
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