TY - JOUR
T1 - Analysis of insertion into secondary attachment sites by phage λ and by int mutants with altered recombination specificity
AU - Rutkai, Edit
AU - Dorgai, László
AU - Sirot, Regina
AU - Yagil, Ezra
AU - Weisberg, Robert A.
N1 - Funding Information:
We are grateful to Xiao Ma and Tom Schneider for helping us to analyze sequence conservation in secondary sites, to Art Landy for his comments on the manuscript, and to Allan Campbell and Howard Nash for helpful discussions and the communication of unpublished information. This work was supported, in part, by an NIH FIRCA grant. E.R. received a PhD fellowship grant from the Bay Zoltán Foundation for Applied Research, E.Y. was supported by the Israel Science Foundation (grant no. 405/98), and the Hungarian Ministry of Education and the Israel Ministry of Science supported travel grants to E.R., L.D., and E.Y.
PY - 2003/6/20
Y1 - 2003/6/20
N2 - When phage λ lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. Here, we characterize the secondary sites that are preferred by wild-type λ and by λ int mutants with altered insertion specificity. The sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. The imperfect inverted repeats of the primary site bind integrase, while the 7 bp spacer, or overlap region, swaps strands with a complementary sequence in the phage attachment site during recombination. We found substantial sequence conservation in the imperfect inverted repeats of secondary sites, and nearly perfect conservation in the leftmost three bases of the overlap region. By contrast, the rightmost bases of the overlap region were much more variable. A phage with an altered overlap region preferred to insert into secondary sites with the corresponding bases. We suggest that this difference between the left and right segments is a result of the defined order of strand exchanges during integrase-promoted recombination. This suggestion accounts for the unexpected segregation pattern of the overlap region observed after insertion into several secondary sites. Some of the altered specificity int mutants differed from wild-type in secondary site preference, but we were unable to identify simple sequence motifs that account for these differences. We propose that insertion into secondary sites is a step in the evolutionary change of phage insertion specificity and present a model of how this might occur.
AB - When phage λ lysogenizes a cell that lacks the primary bacterial attachment site, integrase catalyzes insertion of the phage chromosome into one of many secondary sites. Here, we characterize the secondary sites that are preferred by wild-type λ and by λ int mutants with altered insertion specificity. The sequences of these secondary sites resembled that of the primary site: they contained two imperfect inverted repeats flanking a short spacer. The imperfect inverted repeats of the primary site bind integrase, while the 7 bp spacer, or overlap region, swaps strands with a complementary sequence in the phage attachment site during recombination. We found substantial sequence conservation in the imperfect inverted repeats of secondary sites, and nearly perfect conservation in the leftmost three bases of the overlap region. By contrast, the rightmost bases of the overlap region were much more variable. A phage with an altered overlap region preferred to insert into secondary sites with the corresponding bases. We suggest that this difference between the left and right segments is a result of the defined order of strand exchanges during integrase-promoted recombination. This suggestion accounts for the unexpected segregation pattern of the overlap region observed after insertion into several secondary sites. Some of the altered specificity int mutants differed from wild-type in secondary site preference, but we were unable to identify simple sequence motifs that account for these differences. We propose that insertion into secondary sites is a step in the evolutionary change of phage insertion specificity and present a model of how this might occur.
KW - Evolution
KW - Lysogeny
KW - Phage HK022
KW - Site-specific recombination
KW - Tyrosine recombinase
UR - http://www.scopus.com/inward/record.url?scp=0038206481&partnerID=8YFLogxK
U2 - 10.1016/S0022-2836(03)00442-X
DO - 10.1016/S0022-2836(03)00442-X
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AN - SCOPUS:0038206481
SN - 0022-2836
VL - 329
SP - 983
EP - 996
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -