Limits...
DNA binding and synapsis by the large C-terminal domain of phiC31 integrase.

McEwan AR, Rowley PA, Smith MC - Nucleic Acids Res. (2009)

Bottom Line: Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR).Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex.Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD-CTD interactions and led to defective DNA binding and no detectable DNA synapsis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2BX, UK.

ABSTRACT
The integrase (Int) from phage C31 acts on the phage and host-attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. Excision (attL x attR recombination) is prevented, in the absence of accessory factors, by a putative coiled-coil motif in the C-terminal domain (CTD). Int has a serine recombinase N-terminal domain, required for synapsis of recombination substrates and catalysis. We show here that the coiled-coil motif mediates protein-protein interactions between CTDs, but only when bound to DNA. Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR). Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex. Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD-CTD interactions and led to defective DNA binding and no detectable DNA synapsis. A substitution, E449K, in full length Int confers the ability to perform excision in addition to integration as it has gained the ability to synapse attL x attR. hCTD(E449K) was similar to hCTD in DNA binding but unable to form the CTD synapse suggesting that the CTD synapse is not essential but could be part of the mechanism that controls directionality.

Show MeSH

Related in: MedlinePlus

Model for synapsis and recombination by ϕC31 Int. The substrates attP and attB are shown as grey and black lines. Int subunits bound to a half site derived from attB and attP are shown in blue and red, respectively, and are meant to suggest subtle differences in conformation. Int subunits bound to attP are also shown spaced wider apart than Int subunits bound to attB. Three functional motifs are indicated within each Int subunit as described in the Key. (A) (i) Coiled-coil interactions occur between Int subunits bound to attB but not when bound to attP. (ii) The free coiled coil motifs in attP could interact with the CTD of Int subunits bound to attB to form a CTD synapse containing a tetramer of CTD domains. (iii) We propose that the coiled-coil motif blocks the formation of the NTD synaptic interface and this inhibition must be removed for the formation of a productive synapse. (iv) and (v) Formation of the productive synapse then triggers the DNA cleavage and strand exchange activities of the NTD which occur by a mechanism that resembles that of the resolvase/invertases (11). (vi) After religation of the DNA we propose that there are further conformation changes that establish the coiled-coil interaction once more between adjacently bound Int subunits, now bound to attL and attR. The role, if any, of the coiled-coil motif in the full synapse or during catalysis and strand exchange is not known. (B) A model for synapsis by IntE449K. (i) The coiled-coil motif is shown in an aberrant position where its ability to block oligomerization through the NTDs is reduced. The net result is formation of the full synapse without forming the CTD synapse. (ii) In an attL × attR reaction, we propose that weak CTD:CTD interactions between adjacently bound subunits results in a loss of inhibition on the oligomerization activity of the NTDs, which can lead to formation of a productive synapse. This model shows how the asymmetric binding by Int to attL and attR could lead to the observed preference for complementary subunit interactions at synapsis (as shown here) rather than non-complementary interactions (see text for more details).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2724291&req=5

Figure 6: Model for synapsis and recombination by ϕC31 Int. The substrates attP and attB are shown as grey and black lines. Int subunits bound to a half site derived from attB and attP are shown in blue and red, respectively, and are meant to suggest subtle differences in conformation. Int subunits bound to attP are also shown spaced wider apart than Int subunits bound to attB. Three functional motifs are indicated within each Int subunit as described in the Key. (A) (i) Coiled-coil interactions occur between Int subunits bound to attB but not when bound to attP. (ii) The free coiled coil motifs in attP could interact with the CTD of Int subunits bound to attB to form a CTD synapse containing a tetramer of CTD domains. (iii) We propose that the coiled-coil motif blocks the formation of the NTD synaptic interface and this inhibition must be removed for the formation of a productive synapse. (iv) and (v) Formation of the productive synapse then triggers the DNA cleavage and strand exchange activities of the NTD which occur by a mechanism that resembles that of the resolvase/invertases (11). (vi) After religation of the DNA we propose that there are further conformation changes that establish the coiled-coil interaction once more between adjacently bound Int subunits, now bound to attL and attR. The role, if any, of the coiled-coil motif in the full synapse or during catalysis and strand exchange is not known. (B) A model for synapsis by IntE449K. (i) The coiled-coil motif is shown in an aberrant position where its ability to block oligomerization through the NTDs is reduced. The net result is formation of the full synapse without forming the CTD synapse. (ii) In an attL × attR reaction, we propose that weak CTD:CTD interactions between adjacently bound subunits results in a loss of inhibition on the oligomerization activity of the NTDs, which can lead to formation of a productive synapse. This model shows how the asymmetric binding by Int to attL and attR could lead to the observed preference for complementary subunit interactions at synapsis (as shown here) rather than non-complementary interactions (see text for more details).

Mentions: When ϕC31 Int binds to its attachment sites two complexes are observed (7) (Figures 4G, 5A and D). As free Int is a dimer, we propose that the more abundant and lower mobility complex contains a DNA bound dimer, with each Int subunit contacting one of the two-half sites of each attachment site (7) (Figure 6). Complex I, which is always in much lower abundance and has a higher mobility than complex II, most likely contains a monomer of Int bound to one of the two-half sites (7). When the hCTD was used in binding assays with the attachment sites low (complex II) and high (complex I) mobility complexes were also observed (Figure 4). The binding affinities for the four attachment sites by the hCTD were similar to those obtained with wild-type Int (Figure 4) (7,13). As hCTD was monomeric in solution, we expected the hCTD to occupy the two half sites independently on the basis of concentration and affinity. This appeared to be the case for attP binding where complex I is the most abundant complex up to 83 nM (Figure 4C and D). In contrast the hCTD appeared to bind to attB, attL and attR cooperatively, where even at low concentrations of protein the majority of bound probe was in complex II (Figure 4A, B, E and F). Indeed, binding by the hCTD to attB appeared to be dependent on co-operativity as when a probe (BX50), containing an attB site in which one arm was heavily mutated to prevent binding, was used in a binding assay, the binding affinity by hCTD was severely reduced compared to an attB50 probe (Figure 4G). Under the same conditions the affinity of native Int for BX50 was only mildly affected (Figure 4G). The cooperative binding by hCTD to attB, attL and attR could be explained if protein–protein interactions occurred between adjacently bound hCTDs or if binding to a half site by hCTD altered the DNA conformation to favour binding of a second molecule to the adjacent half site.Figure 4.


DNA binding and synapsis by the large C-terminal domain of phiC31 integrase.

McEwan AR, Rowley PA, Smith MC - Nucleic Acids Res. (2009)

Model for synapsis and recombination by ϕC31 Int. The substrates attP and attB are shown as grey and black lines. Int subunits bound to a half site derived from attB and attP are shown in blue and red, respectively, and are meant to suggest subtle differences in conformation. Int subunits bound to attP are also shown spaced wider apart than Int subunits bound to attB. Three functional motifs are indicated within each Int subunit as described in the Key. (A) (i) Coiled-coil interactions occur between Int subunits bound to attB but not when bound to attP. (ii) The free coiled coil motifs in attP could interact with the CTD of Int subunits bound to attB to form a CTD synapse containing a tetramer of CTD domains. (iii) We propose that the coiled-coil motif blocks the formation of the NTD synaptic interface and this inhibition must be removed for the formation of a productive synapse. (iv) and (v) Formation of the productive synapse then triggers the DNA cleavage and strand exchange activities of the NTD which occur by a mechanism that resembles that of the resolvase/invertases (11). (vi) After religation of the DNA we propose that there are further conformation changes that establish the coiled-coil interaction once more between adjacently bound Int subunits, now bound to attL and attR. The role, if any, of the coiled-coil motif in the full synapse or during catalysis and strand exchange is not known. (B) A model for synapsis by IntE449K. (i) The coiled-coil motif is shown in an aberrant position where its ability to block oligomerization through the NTDs is reduced. The net result is formation of the full synapse without forming the CTD synapse. (ii) In an attL × attR reaction, we propose that weak CTD:CTD interactions between adjacently bound subunits results in a loss of inhibition on the oligomerization activity of the NTDs, which can lead to formation of a productive synapse. This model shows how the asymmetric binding by Int to attL and attR could lead to the observed preference for complementary subunit interactions at synapsis (as shown here) rather than non-complementary interactions (see text for more details).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724291&req=5

Figure 6: Model for synapsis and recombination by ϕC31 Int. The substrates attP and attB are shown as grey and black lines. Int subunits bound to a half site derived from attB and attP are shown in blue and red, respectively, and are meant to suggest subtle differences in conformation. Int subunits bound to attP are also shown spaced wider apart than Int subunits bound to attB. Three functional motifs are indicated within each Int subunit as described in the Key. (A) (i) Coiled-coil interactions occur between Int subunits bound to attB but not when bound to attP. (ii) The free coiled coil motifs in attP could interact with the CTD of Int subunits bound to attB to form a CTD synapse containing a tetramer of CTD domains. (iii) We propose that the coiled-coil motif blocks the formation of the NTD synaptic interface and this inhibition must be removed for the formation of a productive synapse. (iv) and (v) Formation of the productive synapse then triggers the DNA cleavage and strand exchange activities of the NTD which occur by a mechanism that resembles that of the resolvase/invertases (11). (vi) After religation of the DNA we propose that there are further conformation changes that establish the coiled-coil interaction once more between adjacently bound Int subunits, now bound to attL and attR. The role, if any, of the coiled-coil motif in the full synapse or during catalysis and strand exchange is not known. (B) A model for synapsis by IntE449K. (i) The coiled-coil motif is shown in an aberrant position where its ability to block oligomerization through the NTDs is reduced. The net result is formation of the full synapse without forming the CTD synapse. (ii) In an attL × attR reaction, we propose that weak CTD:CTD interactions between adjacently bound subunits results in a loss of inhibition on the oligomerization activity of the NTDs, which can lead to formation of a productive synapse. This model shows how the asymmetric binding by Int to attL and attR could lead to the observed preference for complementary subunit interactions at synapsis (as shown here) rather than non-complementary interactions (see text for more details).
Mentions: When ϕC31 Int binds to its attachment sites two complexes are observed (7) (Figures 4G, 5A and D). As free Int is a dimer, we propose that the more abundant and lower mobility complex contains a DNA bound dimer, with each Int subunit contacting one of the two-half sites of each attachment site (7) (Figure 6). Complex I, which is always in much lower abundance and has a higher mobility than complex II, most likely contains a monomer of Int bound to one of the two-half sites (7). When the hCTD was used in binding assays with the attachment sites low (complex II) and high (complex I) mobility complexes were also observed (Figure 4). The binding affinities for the four attachment sites by the hCTD were similar to those obtained with wild-type Int (Figure 4) (7,13). As hCTD was monomeric in solution, we expected the hCTD to occupy the two half sites independently on the basis of concentration and affinity. This appeared to be the case for attP binding where complex I is the most abundant complex up to 83 nM (Figure 4C and D). In contrast the hCTD appeared to bind to attB, attL and attR cooperatively, where even at low concentrations of protein the majority of bound probe was in complex II (Figure 4A, B, E and F). Indeed, binding by the hCTD to attB appeared to be dependent on co-operativity as when a probe (BX50), containing an attB site in which one arm was heavily mutated to prevent binding, was used in a binding assay, the binding affinity by hCTD was severely reduced compared to an attB50 probe (Figure 4G). Under the same conditions the affinity of native Int for BX50 was only mildly affected (Figure 4G). The cooperative binding by hCTD to attB, attL and attR could be explained if protein–protein interactions occurred between adjacently bound hCTDs or if binding to a half site by hCTD altered the DNA conformation to favour binding of a second molecule to the adjacent half site.Figure 4.

Bottom Line: Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR).Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex.Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD-CTD interactions and led to defective DNA binding and no detectable DNA synapsis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2BX, UK.

ABSTRACT
The integrase (Int) from phage C31 acts on the phage and host-attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. Excision (attL x attR recombination) is prevented, in the absence of accessory factors, by a putative coiled-coil motif in the C-terminal domain (CTD). Int has a serine recombinase N-terminal domain, required for synapsis of recombination substrates and catalysis. We show here that the coiled-coil motif mediates protein-protein interactions between CTDs, but only when bound to DNA. Although the histidine-tagged CTD (hCTD) was monomeric in solution, hCTD bound cooperatively to three of the recombination substrates (attB, attL and attR). Furthermore, when provided with attP and attB, hCTD brought these substrates together in a synaptic complex. Substitutions in the coiled-coil motif that greatly reduce Int integration activity, L460P and Y475H, prevented CTD-CTD interactions and led to defective DNA binding and no detectable DNA synapsis. A substitution, E449K, in full length Int confers the ability to perform excision in addition to integration as it has gained the ability to synapse attL x attR. hCTD(E449K) was similar to hCTD in DNA binding but unable to form the CTD synapse suggesting that the CTD synapse is not essential but could be part of the mechanism that controls directionality.

Show MeSH
Related in: MedlinePlus