TY - JOUR
T1 - Identifying determinants of recombination specificity
T2 - Construction and characterization of mutant bacteriophage integrases
AU - Dorgai, László
AU - Yagil, Ezra
AU - Weisberg, Robert A.
N1 - Funding Information:
This work was supported in part by grant no. 90-349/3 from the United States–Israel Binational Science Foundation (BSF) Jerusalem, Israel, and by grant no. 3297 from the Israel Ministry of Science and Technology. We are grateful to Dr Howard Nash for his comments on the manuscript.
PY - 1995/9/15
Y1 - 1995/9/15
N2 - The Integrases of bacteriophages λ and HK022 promote recombination between DNA molecules that carry attachment sites. The two integrases are about 70% identical in sequence and catalyze nearly identical reactions, but recognize different sets of sites. To identify the amino acids that determine this difference in specificity, we selected mutants of λ integrase with increased ability to recombine HK022 sites. This selection yielded eleven different amino acid substitutions at eight different positions. Three of the positions belong to a larger set that were identified as important for the λ/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK022 for the corresponding λ residue at each of these three positions increased recombination of HK022 sites, and one double substitution, N99D-E319R, increased recombination to nearly wild-type HK022 levels. Mutations at the other five positions changed residues that are identical in the wild-type proteins or are at positions identified by chimera analysis as unimportant for the λ/HK022 specificity difference. All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine λ sites. However, we found that substitution of HK022 for λ residues at three additional positions, S282P, G283K, and R287K, specifically reduced recombination of λ sites. These three substitutions when combined with N99D and E319R were sufficient to change the specificity of λ to that of HK022 integrase. The first three substitutions act principally to prevent recombination of λ sites, and the second two to remove a barrier to recombination of HK022 sites. We suggest that many natural alterations in the specificity of protein-DNA interactions occur by multi-step changes that first relax and then restrict specificity.
AB - The Integrases of bacteriophages λ and HK022 promote recombination between DNA molecules that carry attachment sites. The two integrases are about 70% identical in sequence and catalyze nearly identical reactions, but recognize different sets of sites. To identify the amino acids that determine this difference in specificity, we selected mutants of λ integrase with increased ability to recombine HK022 sites. This selection yielded eleven different amino acid substitutions at eight different positions. Three of the positions belong to a larger set that were identified as important for the λ/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK022 for the corresponding λ residue at each of these three positions increased recombination of HK022 sites, and one double substitution, N99D-E319R, increased recombination to nearly wild-type HK022 levels. Mutations at the other five positions changed residues that are identical in the wild-type proteins or are at positions identified by chimera analysis as unimportant for the λ/HK022 specificity difference. All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine λ sites. However, we found that substitution of HK022 for λ residues at three additional positions, S282P, G283K, and R287K, specifically reduced recombination of λ sites. These three substitutions when combined with N99D and E319R were sufficient to change the specificity of λ to that of HK022 integrase. The first three substitutions act principally to prevent recombination of λ sites, and the second two to remove a barrier to recombination of HK022 sites. We suggest that many natural alterations in the specificity of protein-DNA interactions occur by multi-step changes that first relax and then restrict specificity.
KW - Attachment sites
KW - Integrase
KW - Lambda
KW - Recombination specificity
UR - http://www.scopus.com/inward/record.url?scp=0029147088&partnerID=8YFLogxK
U2 - 10.1006/jmbi.1995.0486
DO - 10.1006/jmbi.1995.0486
M3 - ???researchoutput.researchoutputtypes.contributiontojournal.article???
AN - SCOPUS:0029147088
SN - 0022-2836
VL - 252
SP - 178
EP - 188
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -