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Thermoplasma acidophilum Cdc6 protein stimulates MCM helicase activity by regulating its ATPase activity.

Haugland GT, Sakakibara N, Pey AL, Rollor CR, Birkeland NK, Kelman Z - Nucleic Acids Res. (2008)

Bottom Line: It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity.It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM.The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Bergen, N-5020 Bergen, Norway.

ABSTRACT
The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. In most archaeal species studied, the interaction between MCM and the initiator protein, Cdc6, inhibits helicase activity. To date, the only exception is the helicase and Cdc6 proteins from the archaeon Thermoplasma acidophilum. It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity. It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM. Also, through the use of site-specific substitutions, and truncated and chimeric proteins, it was shown that an intact Cdc6 protein is required for this stimulation. ATP binding and hydrolysis by the Cdc6 protein is not needed for the stimulation. The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity.

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The T. acidophilum Cdc6-2 protein interacts with MCM. Two-hybrid analysis was performed as described in Material and methods section. Cell growth observed after 24 h is marked as ++++, +++ after 48 h and ++ after 72 h.
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Figure 3: The T. acidophilum Cdc6-2 protein interacts with MCM. Two-hybrid analysis was performed as described in Material and methods section. Cell growth observed after 24 h is marked as ++++, +++ after 48 h and ++ after 72 h.

Mentions: It was previously shown that the MCM protein interacts with Cdc6-2 (5). The data presented in Figure 4 demonstrate that only the intact Cdc6-2 proteins can stimulate helicase activity; neither the WH nor the AAA+ domains can stimulate the helicase. Thus, the data may suggest that only the full-length Cdc6-2 protein can bind MCM protein. This is not the case, however. Two-hybrid analysis demonstrated that both the full-length and the AAA+ catalytic portion of Cdc6-2 could interact with MCM (Figure 3). However, no interaction between Cdc6-2 protein and either the N- or C-terminal part of MCM could be detected. Although the AAA+ domains of Cdc6-2 interact with MCM, helicase activity is not stimulated (Figure 4). This suggests that interaction with the helicase is not sufficient to stimulate its activity. These results are reminiscent of those observed with the proteins from M. thermautotrophicus. In M. thermautotrophicus, the interaction between Cdc6 and MCM inhibits helicase activity (20). However, it was shown that although the WH motif of Cdc6 can bind MCM it is not sufficient for helicase inhibition (21).Figure 3.


Thermoplasma acidophilum Cdc6 protein stimulates MCM helicase activity by regulating its ATPase activity.

Haugland GT, Sakakibara N, Pey AL, Rollor CR, Birkeland NK, Kelman Z - Nucleic Acids Res. (2008)

The T. acidophilum Cdc6-2 protein interacts with MCM. Two-hybrid analysis was performed as described in Material and methods section. Cell growth observed after 24 h is marked as ++++, +++ after 48 h and ++ after 72 h.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553600&req=5

Figure 3: The T. acidophilum Cdc6-2 protein interacts with MCM. Two-hybrid analysis was performed as described in Material and methods section. Cell growth observed after 24 h is marked as ++++, +++ after 48 h and ++ after 72 h.
Mentions: It was previously shown that the MCM protein interacts with Cdc6-2 (5). The data presented in Figure 4 demonstrate that only the intact Cdc6-2 proteins can stimulate helicase activity; neither the WH nor the AAA+ domains can stimulate the helicase. Thus, the data may suggest that only the full-length Cdc6-2 protein can bind MCM protein. This is not the case, however. Two-hybrid analysis demonstrated that both the full-length and the AAA+ catalytic portion of Cdc6-2 could interact with MCM (Figure 3). However, no interaction between Cdc6-2 protein and either the N- or C-terminal part of MCM could be detected. Although the AAA+ domains of Cdc6-2 interact with MCM, helicase activity is not stimulated (Figure 4). This suggests that interaction with the helicase is not sufficient to stimulate its activity. These results are reminiscent of those observed with the proteins from M. thermautotrophicus. In M. thermautotrophicus, the interaction between Cdc6 and MCM inhibits helicase activity (20). However, it was shown that although the WH motif of Cdc6 can bind MCM it is not sufficient for helicase inhibition (21).Figure 3.

Bottom Line: It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity.It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM.The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Bergen, N-5020 Bergen, Norway.

ABSTRACT
The minichromosome maintenance (MCM) proteins are thought to function as the replicative helicases in archaea. In most archaeal species studied, the interaction between MCM and the initiator protein, Cdc6, inhibits helicase activity. To date, the only exception is the helicase and Cdc6 proteins from the archaeon Thermoplasma acidophilum. It was previously shown that when the Cdc6 protein interacts with MCM it substantially stimulates helicase activity. It is shown here that the mechanism by which the Cdc6 protein stimulates helicase activity is by stimulating the ATPase activity of MCM. Also, through the use of site-specific substitutions, and truncated and chimeric proteins, it was shown that an intact Cdc6 protein is required for this stimulation. ATP binding and hydrolysis by the Cdc6 protein is not needed for the stimulation. The data suggest that binding of Cdc6 protein to MCM protein changes the structure of the helicase, enhancing the catalytic hydrolysis of ATP and helicase activity.

Show MeSH