A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination
Nucleic Acids Research
A motif in the C-terminal domain of rC31 integrase controls the directionality of recombination
Paul A. Rowley 0
Matthew C. A. Smith 1
Ellen Younger 0
Margaret C. M. Smith 0
0 Institute of Medical Sciences, University of Aberdeen , Foresterhill, Aberdeen AB252ZD
1 Institute of Genetics, University of Nottingham , Nottingham NG7 2UH , UK
Bacteriophage rC31 encodes an integrase, which acts on the phage and host attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. In the absence of accessory factors, rC31 integrase cannot catalyse attL x attR recombination to excise the prophage. To understand the mechanism of directionality, mutant integrases were characterized that were active in excision. A hyperactive integrase, Int E449K, gained the ability to catalyse attL x attR, attL x attL and attR x attR recombination whilst retaining the ability to recombine attP x attB. A catalytically defective derivative of this mutant, Int S12A, E449K, could form stable complexes with attP/attB, attL/attR, attL/attL and attR/attR under conditions where Int S12A only complexed with attP/attB. Further analysis of the Int E449K-attL/attR synaptic events revealed a preference for one of the two predicted synapse structures with different orientations of the attL/attR sites. Several amino acid substitutions conferring hyperactivity, including E449K, were localized to one face of a predicted coiled-coil motif in the C-terminal domain. This work shows that a motif in the C-terminal domain of rC31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein-protein interactions.
INTRODUCTION
In order to establish a lysogenic life style, many
bacteriophages encode an integrase to integrate the
phage genome into the host chromosome (
1,2
). In
association with an accessory factor, the recombination
directionality factor (RDF) or Xis, the integrase also
excises the phage genome on induction into the lytic
pathway (
2,3
). During integration, the phage integrases
act site-specifically by recombining the host and phage
attachment sites, attB and attP, to form the hybrid
products attL and attR. In excision attL and attR
recombine to regenerate attB and attP. Phage integration
and excision has to be a highly regulated process to ensure
efficient switching between the lytic pathway and lysogeny
(
1
). As some integrases are unidirectional in the absence
of accessory factors, phage integrases can be used for
site-specific genome manipulations and for the stable
integration of transgenes (
4
). The integrase from the
Streptomyces temperate phage fC31 is currently the most
widely used phage integrase for genome manipulations
(
5?14
).
The fC31 integrase is a member of a large family of
site-specific recombinases, the serine recombinases (
15,16
).
The best understood members of this family are the
resolvase/invertases, in particular gd and Tn3 resolvases
and Hin invertase. The resolvase/invertases have an
N-terminal catalytic domain ( 1?140 aa) and a small
( 141?183 aa), C-terminal DNA-binding domain. The
fC31 integrase is a member of the large serine
recombinases (17). These proteins have similar N-terminal
catalytic domains to those of the resolvase/invertases,
but they have much larger C-terminal domains of 300?
500 aa. The large serine recombinases also include some
transposases such as TnpX from the Clostridium
perfringens transposon, Tn4451, and TndX from the C. difficile
conjugative transposon CTn5397 (
18?20
). Tn4451 and
CTn5397 confer resistances to chloramphenicol and
tetracycline, respectively. TnpX and TndX have been
shown to be capable of integrating and excising their
respective transposons with no requirement for any
additional transposon-encoded accessory factors (
20,21
).
The first step in a site-specific recombination pathway
is recognition and binding of the substrates by the
recombinase (
22
). Protein?protein interactions between
the recombinase subunits then bring the two substrates
together in a synaptic complex. Ghosh et al. (
23
) showed
that dimers of mycobacteriophage Bxb1 integrase (a large
serine recombinase) bind the substrates attB and attP,
which are then probably brought together to form a
synaptic tetramer. Post-synapsis, the resolvase/invertases and
the large serine recombinases appear to use the same
catalytic mechanism (
16,22?27
). DNA cleavage generates
2 bp staggered breaks in both substrates with the
concomitant formation of transient covalent
phosphoserine bonds between the recombinases and the recessed
50 ends. Strand exchange is thought to occur by 1808
rotation of two recombinase subunits bound to half sites
relative to the other two subunits (
28?30
). Joining of the
half sites to form recombinant products is inhibited when
the two base overhangs at the staggered breaks cannot
base pair due to mismatches (
31,32
). In the serine
integras (...truncated)