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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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Size exclusion chromatography of the isolated hCTD and mutant derivatives. Gel filtration traces of hCTD (green), hCTDY475H (grey), hCTDL460P (red) and hCTDE449K (light blue) and wild-type Int (black). The calculated momomeric form of the hCTD is 54 kDa. The apparent molecular weights for hCTD, hCTDE449K, hCTDL460P and hCTDY475H were 52 kDa, 79 kDa, 84 kDa and 55 kDa, respectively. Full length Int eluted with an apparent molecular weight of 121 kDa (predicted monomer is 67 kDa).
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Figure 3: Size exclusion chromatography of the isolated hCTD and mutant derivatives. Gel filtration traces of hCTD (green), hCTDY475H (grey), hCTDL460P (red) and hCTDE449K (light blue) and wild-type Int (black). The calculated momomeric form of the hCTD is 54 kDa. The apparent molecular weights for hCTD, hCTDE449K, hCTDL460P and hCTDY475H were 52 kDa, 79 kDa, 84 kDa and 55 kDa, respectively. Full length Int eluted with an apparent molecular weight of 121 kDa (predicted monomer is 67 kDa).

Mentions: In size exclusion chromatography the hCTD behaved as a monomer, eluting as a 52-kDa protein (Figure 3). The monomeric nature of the hCTD is consistent with the observation that the isolated CTD from the related serine Int from phage Bxb1 is also monomeric (6). Amino-acid substitutions E449K, L460P and Y475H were introduced into hCTD. hCTDY475H behaved in a similar manner to the wild-type hCTD in size exclusion chromatography, whilst hCTDE449K and hCTDL460P had predicted molecular weights of 79 and 84 kDa, respectively (Figure 3). The lower retention volumes observed with hCTDE449K and hCTDL460P most likely reflect differences in protein conformation rather than the acquisition of an oligomerization interface as full length, IntL460P and IntY475H were indistinguishable from native Int in size exclusion chromatography (13). These data suggest that the putative coiled-coil motif is either buried or sequestered when Int is free in solution.Figure 3.


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

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

Size exclusion chromatography of the isolated hCTD and mutant derivatives. Gel filtration traces of hCTD (green), hCTDY475H (grey), hCTDL460P (red) and hCTDE449K (light blue) and wild-type Int (black). The calculated momomeric form of the hCTD is 54 kDa. The apparent molecular weights for hCTD, hCTDE449K, hCTDL460P and hCTDY475H were 52 kDa, 79 kDa, 84 kDa and 55 kDa, respectively. Full length Int eluted with an apparent molecular weight of 121 kDa (predicted monomer is 67 kDa).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Size exclusion chromatography of the isolated hCTD and mutant derivatives. Gel filtration traces of hCTD (green), hCTDY475H (grey), hCTDL460P (red) and hCTDE449K (light blue) and wild-type Int (black). The calculated momomeric form of the hCTD is 54 kDa. The apparent molecular weights for hCTD, hCTDE449K, hCTDL460P and hCTDY475H were 52 kDa, 79 kDa, 84 kDa and 55 kDa, respectively. Full length Int eluted with an apparent molecular weight of 121 kDa (predicted monomer is 67 kDa).
Mentions: In size exclusion chromatography the hCTD behaved as a monomer, eluting as a 52-kDa protein (Figure 3). The monomeric nature of the hCTD is consistent with the observation that the isolated CTD from the related serine Int from phage Bxb1 is also monomeric (6). Amino-acid substitutions E449K, L460P and Y475H were introduced into hCTD. hCTDY475H behaved in a similar manner to the wild-type hCTD in size exclusion chromatography, whilst hCTDE449K and hCTDL460P had predicted molecular weights of 79 and 84 kDa, respectively (Figure 3). The lower retention volumes observed with hCTDE449K and hCTDL460P most likely reflect differences in protein conformation rather than the acquisition of an oligomerization interface as full length, IntL460P and IntY475H were indistinguishable from native Int in size exclusion chromatography (13). These data suggest that the putative coiled-coil motif is either buried or sequestered when Int is free in solution.Figure 3.

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