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Characterization of the Cdc6 Homologues from the Euryarchaeon Thermoplasma acidophilum.

Haugland GT, Innselset M, Madern D, Birkeland NK - Open Biochem J (2008)

Bottom Line: In the present study, a biochemical characterization of the two Cdc6 proteins from the archaeon Thermoplasma acidophilum has been performed.Both TaCdc6-1 and TaCdc6-2 behave as monomers in solution and both are abundantly expressed in vivo.Further, TaCdc6-1 shows strong ability to undergo autophosphorylation compared to TaCdc6-2 and the autophosphorylation activity is not affected by DNA or MCM.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Bergen, P.O. Box 7800, N-5020 Bergen, Norway.

ABSTRACT
Archaeal cell division cycle protein 6 (Cdc6) homologues are thought to be involved in the initiation process of DNA replication. In the present study, a biochemical characterization of the two Cdc6 proteins from the archaeon Thermoplasma acidophilum has been performed. Both TaCdc6-1 and TaCdc6-2 behave as monomers in solution and both are abundantly expressed in vivo. Further, TaCdc6-1 shows strong ability to undergo autophosphorylation compared to TaCdc6-2 and the autophosphorylation activity is not affected by DNA or MCM.

No MeSH data available.


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A. The activity of TaCdc6 is not affected by DNA. Cdc6 autophosphorylation reactions were performed as described in “Materials and Methods”in a reaction mixture (15 µl) containing 10 pmol Cdc6 protein in the absence (lane 2 and 5) or in the presence of 1 µg ssDNA (lane 3 and 6) or 1 µg dsDNA ((lane 4 and 7). Upper panel shows a representative 12% Coomassie stained SDS-polyacrylamide gel. Lower panel shows a representative autoradiogram.B. Cdc6 autophosphorylation reactions were performed as described in “Materials and Methods” in a reaction mixture (15 µl) containing 10 pmol Cdc6 protein in the absence (lane 3 and 5) or presence of 20 pmol MCM (lane 4 and 6). Upper panel shows a representative 12% Coomassie stained SDS-polyacrylamide gel. Lower panel shows a representative autoradiogram.
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Figure 4: A. The activity of TaCdc6 is not affected by DNA. Cdc6 autophosphorylation reactions were performed as described in “Materials and Methods”in a reaction mixture (15 µl) containing 10 pmol Cdc6 protein in the absence (lane 2 and 5) or in the presence of 1 µg ssDNA (lane 3 and 6) or 1 µg dsDNA ((lane 4 and 7). Upper panel shows a representative 12% Coomassie stained SDS-polyacrylamide gel. Lower panel shows a representative autoradiogram.B. Cdc6 autophosphorylation reactions were performed as described in “Materials and Methods” in a reaction mixture (15 µl) containing 10 pmol Cdc6 protein in the absence (lane 3 and 5) or presence of 20 pmol MCM (lane 4 and 6). Upper panel shows a representative 12% Coomassie stained SDS-polyacrylamide gel. Lower panel shows a representative autoradiogram.

Mentions: Protein autophosphorylation activity was measured in reaction mixtures (15 μl) containing 20 mM Hepes-NaOH, pH 7.5, 5 mM MgCl2, 2 mM DTT, 450 ng BSA, 3.3 pmol of [γ-32P]ATP (GE Bioscience), 10 pmol TaCdc6 proteins with or without 1 μg ssDNA (ΦX174 virion DNA; New England Biolabs) or dsDNA (ΦX174 RFII DNA; New England Biolabs) and with or without 20 pmol TaMCM as indicated in the figure legends. Following incubation at 58°C for 20 min, the reaction was stopped by adding 5 μl of 5 x SDS loading buffer (250 mM Tris-HCl, pH 6.8, 500 mM DTT, 10% SDS, 0.5% Bromophenol blue and 50% glycerol). The reaction mixtures were boiled for 5 min and subsequently separated by 12% SDS-PAGE performed at 190 V for 60 min and stained with Coomassie blue. The gel was dried and 32P-labeled bands were detected using phosphorimaging. The experiments were repeated three times. A representative gel and autoradiograph is shown in Fig. (4A and B).


Characterization of the Cdc6 Homologues from the Euryarchaeon Thermoplasma acidophilum.

Haugland GT, Innselset M, Madern D, Birkeland NK - Open Biochem J (2008)

A. The activity of TaCdc6 is not affected by DNA. Cdc6 autophosphorylation reactions were performed as described in “Materials and Methods”in a reaction mixture (15 µl) containing 10 pmol Cdc6 protein in the absence (lane 2 and 5) or in the presence of 1 µg ssDNA (lane 3 and 6) or 1 µg dsDNA ((lane 4 and 7). Upper panel shows a representative 12% Coomassie stained SDS-polyacrylamide gel. Lower panel shows a representative autoradiogram.B. Cdc6 autophosphorylation reactions were performed as described in “Materials and Methods” in a reaction mixture (15 µl) containing 10 pmol Cdc6 protein in the absence (lane 3 and 5) or presence of 20 pmol MCM (lane 4 and 6). Upper panel shows a representative 12% Coomassie stained SDS-polyacrylamide gel. Lower panel shows a representative autoradiogram.
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Figure 4: A. The activity of TaCdc6 is not affected by DNA. Cdc6 autophosphorylation reactions were performed as described in “Materials and Methods”in a reaction mixture (15 µl) containing 10 pmol Cdc6 protein in the absence (lane 2 and 5) or in the presence of 1 µg ssDNA (lane 3 and 6) or 1 µg dsDNA ((lane 4 and 7). Upper panel shows a representative 12% Coomassie stained SDS-polyacrylamide gel. Lower panel shows a representative autoradiogram.B. Cdc6 autophosphorylation reactions were performed as described in “Materials and Methods” in a reaction mixture (15 µl) containing 10 pmol Cdc6 protein in the absence (lane 3 and 5) or presence of 20 pmol MCM (lane 4 and 6). Upper panel shows a representative 12% Coomassie stained SDS-polyacrylamide gel. Lower panel shows a representative autoradiogram.
Mentions: Protein autophosphorylation activity was measured in reaction mixtures (15 μl) containing 20 mM Hepes-NaOH, pH 7.5, 5 mM MgCl2, 2 mM DTT, 450 ng BSA, 3.3 pmol of [γ-32P]ATP (GE Bioscience), 10 pmol TaCdc6 proteins with or without 1 μg ssDNA (ΦX174 virion DNA; New England Biolabs) or dsDNA (ΦX174 RFII DNA; New England Biolabs) and with or without 20 pmol TaMCM as indicated in the figure legends. Following incubation at 58°C for 20 min, the reaction was stopped by adding 5 μl of 5 x SDS loading buffer (250 mM Tris-HCl, pH 6.8, 500 mM DTT, 10% SDS, 0.5% Bromophenol blue and 50% glycerol). The reaction mixtures were boiled for 5 min and subsequently separated by 12% SDS-PAGE performed at 190 V for 60 min and stained with Coomassie blue. The gel was dried and 32P-labeled bands were detected using phosphorimaging. The experiments were repeated three times. A representative gel and autoradiograph is shown in Fig. (4A and B).

Bottom Line: In the present study, a biochemical characterization of the two Cdc6 proteins from the archaeon Thermoplasma acidophilum has been performed.Both TaCdc6-1 and TaCdc6-2 behave as monomers in solution and both are abundantly expressed in vivo.Further, TaCdc6-1 shows strong ability to undergo autophosphorylation compared to TaCdc6-2 and the autophosphorylation activity is not affected by DNA or MCM.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Bergen, P.O. Box 7800, N-5020 Bergen, Norway.

ABSTRACT
Archaeal cell division cycle protein 6 (Cdc6) homologues are thought to be involved in the initiation process of DNA replication. In the present study, a biochemical characterization of the two Cdc6 proteins from the archaeon Thermoplasma acidophilum has been performed. Both TaCdc6-1 and TaCdc6-2 behave as monomers in solution and both are abundantly expressed in vivo. Further, TaCdc6-1 shows strong ability to undergo autophosphorylation compared to TaCdc6-2 and the autophosphorylation activity is not affected by DNA or MCM.

No MeSH data available.


Related in: MedlinePlus