IDENTIFYING DETERMINANTS OF RECOMBINATION SPECIFICITY - CONSTRUCTION AND CHARACTERIZATION OF MUTANT BACTERIOPHAGE INTEGRASES

被引:59
作者
DORGAI, L
YAGIL, E
WEISBERG, RA
机构
[1] NICHHD,GENET MOLEC LAB,MICROBIAL GENET SECT,BETHESDA,MD 20892
[2] TEL AVIV UNIV,GEORGE S WISE FAC LIFE SCI,DEPT BIOCHEM,IL-69978 TEL AVIV,ISRAEL
关键词
LAMBDA; INTEGRASE; RECOMBINATION SPECIFICITY; ATTACHMENT SITES;
D O I
10.1006/jmbi.1995.0486
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
The Integrases of bacteriophages lambda 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 lambda 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 lambda/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK022 for the corresponding lambda 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 lambda/HK022 specificity difference. All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine lambda sites. However, we found that substitution of HK022 for lambda residues at three additional positions, S282P, G283K, and R287X, specifically reduced recombination of lambda sites. These three substitutions when combined with N99D and E319R were sufficient to change the specificity of lambda to that of HK022 integrase. The first three substitutions act principally to prevent recombination of lambda 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 (C) 1995 Academic Press Limited
引用
收藏
页码:178 / 188
页数:11
相关论文
共 29 条
  • [1] CLEAVAGE OF THE SITE-SPECIFIC RECOMBINATION PROTEIN GAMMA-DELTA RESOLVASE - THE SMALLER OF 2 FRAGMENTS BINDS DNA SPECIFICALLY
    ABDELMEGUID, SS
    GRINDLEY, NDF
    TEMPLETON, NS
    STEITZ, TA
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES, 1984, 81 (07): : 2001 - 2005
  • [2] MUTATIONAL ANALYSIS OF THE LAMBDA INT GENE - DNA-SEQUENCE OF DOMINANT MUTATIONS
    BEAR, SE
    CLEMENS, JB
    ENQUIST, LW
    ZAGURSKY, RJ
    [J]. JOURNAL OF BACTERIOLOGY, 1987, 169 (12) : 5880 - 5883
  • [3] AN INTEGRATION-PROFICIENT INT MUTANT OF BACTERIOPHAGE-LAMBDA
    ENQUIST, LW
    WEISBERG, RA
    [J]. MOLECULAR AND GENERAL GENETICS, 1984, 195 (1-2): : 62 - 69
  • [4] ROLE OF LAMBDA-INTEGRASE IN INTEGRATION AND EXCISION
    ENQUIST, LW
    KIKUCHI, A
    WEISBERG, RA
    [J]. COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, 1979, 43 : 1115 - 1120
  • [5] ANALYSIS OF A NUCLEOPROTEIN COMPLEX - THE SYNAPTOSOME OF GAMMA-DELTA RESOLVASE
    GRINDLEY, NDF
    [J]. SCIENCE, 1993, 262 (5134) : 738 - 740
  • [6] DNA RECOGNITION BY PROTEINS WITH THE HELIX-TURN-HELIX MOTIF
    HARRISON, SC
    AGGARWAL, AK
    [J]. ANNUAL REVIEW OF BIOCHEMISTRY, 1990, 59 : 933 - 969
  • [7] LAMBDA-INT PROTEIN BRIDGES BETWEEN HIGHER-ORDER COMPLEXES AT 2 DISTANT CHROMOSOMAL LOCI ATTL AND ATTR
    KIM, S
    LANDY, A
    [J]. SCIENCE, 1992, 256 (5054) : 198 - 203
  • [8] MAPPING OF A HIGHER-ORDER PROTEIN-DNA COMPLEX - 2 KINDS OF LONG-RANGE INTERACTIONS IN LAMBDA-ATTL
    KIM, SH
    DEVARGAS, LM
    NUNESDUBY, SE
    LANDY, A
    [J]. CELL, 1990, 63 (04) : 773 - 781
  • [9] MHHAI BINDS TIGHTLY TO SUBSTRATES CONTAINING MISMATCHES AT THE TARGET BASE
    KLIMASAUSKAS, S
    ROBERTS, RJ
    [J]. NUCLEIC ACIDS RESEARCH, 1995, 23 (08) : 1388 - 1395
  • [10] THE DNA (CYTOSINE-5) METHYLTRANSFERASES
    KUMAR, S
    CHENG, XD
    KLIMASAUSKAS, S
    MI, S
    POSFAI, J
    ROBERTS, RJ
    WILSON, GG
    [J]. NUCLEIC ACIDS RESEARCH, 1994, 22 (01) : 1 - 10