Yagil E, Dorgai L, Weisberg R A
Section on Microbial Genetics, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA.
J Mol Biol. 1995 Sep 15;252(2):163-77. doi: 10.1006/jmbi.1995.0485.
Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages lambda and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment sites. To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of lambda and HK022 sites in vivo. A chimera that is of lambda origin except for one HK022 residue at position 99 and 12 HK022 residues located between positions 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity. The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were inactive in recombining either HK022 or lambda sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.
噬菌体整合酶是一类结构相关的酶家族成员，可促进携带特定位点的DNA分子之间的重组。噬菌体λ和HK022编码密切相关的整合酶，它们识别各自附着位点核心区域内不同的序列集。为了定位决定这种特异性差异的氨基酸残基，我们分离出产生嵌合整合酶的重组噬菌体，并测量这些嵌合体在体内促进λ和HK022位点重组的能力。除了第99位的一个HK022残基以及位于279位和329位之间的12个HK022残基外，一个源自λ的嵌合体具有野生型HK022的特异性以及整合和切除重组的活性。包含这13个残基某些子集的嵌合体具有不完全的特异性。第99位周围的区域在整合酶家族的其他成员中保守性不强，但279 - 329片段包含高度保守且被认为直接参与催化的残基。许多嵌合体在重组HK022或λ位点时无活性。对恢复这些嵌合体活性的突变体进行筛选，揭示了可能相互作用的残基集。
