| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Genetics and Molecular Biology |
ndsteda,1Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark1
Author for correspondence: Karin Hammer. Tel: +45 45 25 24 96. Fax: +45 45 88 26 60. e-mail: karin.hammer{at}biocentrum.dtu.dk
The site-specific recombination system of temperate lactococcal bacteriophage TP901-1 is unusual in several respects. First, the integrase belongs to the family of extended resolvases rather than to the 
integrase family and second, in the presence of this integrase, a 56 bp attP fragment is sufficient for efficient recombination with the chromosomal attB site in the host Lactococcus lactis subsp. cremoris MG1363. In the present work, this attB site was analysed and a 43 bp attB region was found to be the smallest fragment able to participate fully in recombination. In vitro studies showed that the TP901-1 integrase binds this 43 bp attB fragment, the 56 bp attP and a larger attP fragment with equal affinity. Mutational analysis of the 5 bp common core region (TCAAT) showed that the TC dinucleotide is essential for recombination, but not for binding of the integrase, whereas none of the last three bases are important for recombination. When a number of attL sites, obtained by recombination between an attB site containing a mutation in this TC dinucleotide and a wild-type attP site, were sequenced, a mix of sites with the wild-type or the mutated sequence was obtained. These results are consistent with the hypothesis that the TC dinucleotide constitutes the TP901-1 overlap region. A 2 bp overlap region has been observed in recombination reactions catalysed by all other members of the resolvase/invertase family tested so far. By selecting for attB sites with a decreased ability to participate in recombination, two bases located outside the core region of attB were shown to be involved in the in vitro binding of the TP901-1 integrase.
Keywords: lactococcal bacteriophage, extended resolvase, site-specific recombination, mechanism of recombination, chromosomal attachment site
Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Ery, erythromycin; Kn, kanamycin
The GenBank accession number for the sequence reported in this paper is Y15043.
a Present address: Department of Dairy and Food Science, Rolighedsvej 30, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg, Denmark.
This article has been cited by other articles:
|
|
 |
|
  P. Ghosh, L. A. Bibb, and G. F. Hatfull Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading PNAS, March 4, 2008; 105(9): 3238 - 3243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Gupta, R. Till, and M. C. M. Smith Sequences in attB that affect the ability of {phi}C31 integrase to synapse and to activate DNA cleavage Nucleic Acids Res., May 11, 2007; 35(10): 3407 - 3419. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. A. Smith, R. Till, K. Brady, P. Soultanas, H. Thorpe, and M. C. M. Smith Synapsis and DNA cleavage in {phi}C31 integrase-mediated site-specific recombination Nucleic Acids Res., May 11, 2004; 32(8): 2607 - 2617. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Gregory, R. Till, and M. C. M. Smith Integration Site for Streptomyces Phage {phi}BT1 and Development of Site-Specific Integrating Vectors J. Bacteriol., September 1, 2003; 185(17): 5320 - 5323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Stoll, D. S. Ginsburg, and M. P. Calos Phage TP901-1 Site-Specific Integrase Functions in Human Cells J. Bacteriol., July 1, 2002; 184(13): 3657 - 3663. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
