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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.

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The putative coiled-coil motif mediates protein–protein interactions. (A) Gel filtration traces of the hMBP fusions to fragments of Int containing the putative coiled-coil motif. Standards (BioRad) were run before and after the samples. The calculated monomeric molecular weights for hMBP-α, hMBP-β and hMBP-γ were 50 kDa, 50 kDa and 54 kDa, respectively, and the apparent molecular weights were 52 kDa, 36 kDa and 125 kDa, respectively. (B) Gel filtration of hMBP-γ containing mutations which had no effect (hMBP-γE449K) or prevented oligomerization (hMBP-γL460P, hMBP-γY475H and hMBP-γG485E). The apparent molecular weights of these constructs were 125 kDa, 63 kDa, 58 kDa and 67 kDa, respectively. (C and D) Glutaraldehyde crosslinking of purified hMBP and hMBP fusions to fragments containing the putative coiled-coil motif. After treatment with glutaraldehyde the proteins were separated by SDS–PAGE and stained with Coomassie blue. Molecular weight standards were from BioRad.
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Figure 2: The putative coiled-coil motif mediates protein–protein interactions. (A) Gel filtration traces of the hMBP fusions to fragments of Int containing the putative coiled-coil motif. Standards (BioRad) were run before and after the samples. The calculated monomeric molecular weights for hMBP-α, hMBP-β and hMBP-γ were 50 kDa, 50 kDa and 54 kDa, respectively, and the apparent molecular weights were 52 kDa, 36 kDa and 125 kDa, respectively. (B) Gel filtration of hMBP-γ containing mutations which had no effect (hMBP-γE449K) or prevented oligomerization (hMBP-γL460P, hMBP-γY475H and hMBP-γG485E). The apparent molecular weights of these constructs were 125 kDa, 63 kDa, 58 kDa and 67 kDa, respectively. (C and D) Glutaraldehyde crosslinking of purified hMBP and hMBP fusions to fragments containing the putative coiled-coil motif. After treatment with glutaraldehyde the proteins were separated by SDS–PAGE and stained with Coomassie blue. Molecular weight standards were from BioRad.

Mentions: The region 445–524 is predicted to form two α helices H1 and H2 separated by a short linker (Figure 1) (13). Based on a COILS prediction the first of these helices, H1, has a high probability of forming coiled-coil whereas the probability of H2 being coiled-coil was much lower (13,17). Several substitutions in this motif (E449K, E452K, E456K, E463K) were shown previously to cause hyperactivity, allowing these mutant Ints to recombine attL × attR in addition to attP × attB (13). Other mutations (L460P and Y475H) in this putative coiled-coil region were strongly defective in attB × attP recombination (13). Coiled-coil secondary structures often mediate protein–protein interactions and so the ability of the 445–524 regions to oligomerize was investigated. Three constructs were made, hMBP-α, hMBP-β and hMBP-γ, that respectively contained H1, H2 and H1 and H2 fused to a histidine-tagged maltose-binding protein (hMBP). hMBP, hMBP-α and hMBP-β all behaved as monomers by size exclusion chromatography, whereas hMBP-γ behaved as a dimer (Figure 2A). These observations were confirmed by glutaraldehyde crosslinking (Figure 2C). Thus both the predicted H1 and H2 helical regions are required for the efficient dimerization of the motif. The mutations from IntE449K, IntL460P and IntY475H were introduced into hMBP-γ. hMBP-γE449K behaved in a similar manner to hMBP-γ in size exclusion chromatography but hMBP-γY475H and hMBP-γL460P both lost the ability to dimerize (Figure 2B and D). A substitution, G485E, in the putative linker between H1 and H2 was also tested (Figure 1A) as IntG485E was defective for attP × attB recombination but able to recombine attL × attR at very low levels in in vivo recombination assays (data not shown). hMBPγG485E was also unable to dimerize (Figure 2B and D). These data show that, in isolation, the putative coiled-coil motif mediates protein–protein interactions and this interaction is required for Int function.Figure 1.


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

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

The putative coiled-coil motif mediates protein–protein interactions. (A) Gel filtration traces of the hMBP fusions to fragments of Int containing the putative coiled-coil motif. Standards (BioRad) were run before and after the samples. The calculated monomeric molecular weights for hMBP-α, hMBP-β and hMBP-γ were 50 kDa, 50 kDa and 54 kDa, respectively, and the apparent molecular weights were 52 kDa, 36 kDa and 125 kDa, respectively. (B) Gel filtration of hMBP-γ containing mutations which had no effect (hMBP-γE449K) or prevented oligomerization (hMBP-γL460P, hMBP-γY475H and hMBP-γG485E). The apparent molecular weights of these constructs were 125 kDa, 63 kDa, 58 kDa and 67 kDa, respectively. (C and D) Glutaraldehyde crosslinking of purified hMBP and hMBP fusions to fragments containing the putative coiled-coil motif. After treatment with glutaraldehyde the proteins were separated by SDS–PAGE and stained with Coomassie blue. Molecular weight standards were from BioRad.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: The putative coiled-coil motif mediates protein–protein interactions. (A) Gel filtration traces of the hMBP fusions to fragments of Int containing the putative coiled-coil motif. Standards (BioRad) were run before and after the samples. The calculated monomeric molecular weights for hMBP-α, hMBP-β and hMBP-γ were 50 kDa, 50 kDa and 54 kDa, respectively, and the apparent molecular weights were 52 kDa, 36 kDa and 125 kDa, respectively. (B) Gel filtration of hMBP-γ containing mutations which had no effect (hMBP-γE449K) or prevented oligomerization (hMBP-γL460P, hMBP-γY475H and hMBP-γG485E). The apparent molecular weights of these constructs were 125 kDa, 63 kDa, 58 kDa and 67 kDa, respectively. (C and D) Glutaraldehyde crosslinking of purified hMBP and hMBP fusions to fragments containing the putative coiled-coil motif. After treatment with glutaraldehyde the proteins were separated by SDS–PAGE and stained with Coomassie blue. Molecular weight standards were from BioRad.
Mentions: The region 445–524 is predicted to form two α helices H1 and H2 separated by a short linker (Figure 1) (13). Based on a COILS prediction the first of these helices, H1, has a high probability of forming coiled-coil whereas the probability of H2 being coiled-coil was much lower (13,17). Several substitutions in this motif (E449K, E452K, E456K, E463K) were shown previously to cause hyperactivity, allowing these mutant Ints to recombine attL × attR in addition to attP × attB (13). Other mutations (L460P and Y475H) in this putative coiled-coil region were strongly defective in attB × attP recombination (13). Coiled-coil secondary structures often mediate protein–protein interactions and so the ability of the 445–524 regions to oligomerize was investigated. Three constructs were made, hMBP-α, hMBP-β and hMBP-γ, that respectively contained H1, H2 and H1 and H2 fused to a histidine-tagged maltose-binding protein (hMBP). hMBP, hMBP-α and hMBP-β all behaved as monomers by size exclusion chromatography, whereas hMBP-γ behaved as a dimer (Figure 2A). These observations were confirmed by glutaraldehyde crosslinking (Figure 2C). Thus both the predicted H1 and H2 helical regions are required for the efficient dimerization of the motif. The mutations from IntE449K, IntL460P and IntY475H were introduced into hMBP-γ. hMBP-γE449K behaved in a similar manner to hMBP-γ in size exclusion chromatography but hMBP-γY475H and hMBP-γL460P both lost the ability to dimerize (Figure 2B and D). A substitution, G485E, in the putative linker between H1 and H2 was also tested (Figure 1A) as IntG485E was defective for attP × attB recombination but able to recombine attL × attR at very low levels in in vivo recombination assays (data not shown). hMBPγG485E was also unable to dimerize (Figure 2B and D). These data show that, in isolation, the putative coiled-coil motif mediates protein–protein interactions and this interaction is required for Int function.Figure 1.

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