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. 2005 Sep 8;33(15):4940-50.
doi: 10.1093/nar/gki807. Print 2005.

Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

Rajesh Kasiviswanathan  1 , Jae-Ho Shin, Zvi Kelman
Affiliations

Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

Rajesh Kasiviswanathan et al. Nucleic Acids Res. .

Abstract

The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein-protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6-MCM interaction, but not Cdc6-DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.

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Figures

Figure 1
Figure 1
Cdc6-1 protein interacts with MCM protein in a two-hybrid analysis. A summary of the two-hybrid analysis of the interactions between the various M.thermautotrophicus (A) and P.aerophilum (B) MCM- and Cdc6-derived proteins, performed as described in Materials and Methods. In (A), cell growth observed after 24 h, ++++; 48 h, +++; 72 h, ++; 96 h, + and no growth, - are shown. In (B), ‘tick mark’ indicates cell growth after 2 days and ‘cross’ indicates that no growth was observed.
Figure 2
Figure 2
Cdc6 proteins interact with MCM protein in a Far western analysis. A Far western assay was performed as described in Materials and Methods with various concentrations of Cdc6-1 and -2 derived proteins and M.thermautotrophicus 32P-labeled proteins as probes. (A) A schematic representation of the Far western dot blot assay. (B) The Cdc6 and MCM proteins used in the study. ‘X’, in the FLmut and WHmut of Cdc6 in (B), indicates the position of the WH mutations (Cdc6-1, R334,335→A and Cdc6-2, R337→A). (C) A representative blot obtained using FL MCM as a probe. The amount of proteins used in the blot is lanes 1 and 7, 0.05 nmol; lanes 2 and 8, 0.15 nmol; lanes 3 and 9, 0.25 nmol; lanes 4 and 10, 0.5 nmol; lanes 5 and 11, 1.5 nmol; lanes 6 and 12, 2.5 nmol. (D–M) The averages of three independent experiments (with error bars) for the amounts of the various probes used bound to the Cdc6-1 and -2 derived proteins. The colors used are red, FL MCM protein; blue, N-ter MCM; green, ΔA MCM; brown, ΔB MCM; orange, ΔC MCM; gray, PCNA. The colors used are also shown at the bottom of the figure.
Figure 3
Figure 3
Cdc6-1 and -2 proteins interact with MCM in solution. Protein pull-down assays were performed as described in Materials and Methods by binding 2 µg of MBP-tagged Cdc6-1 or -2 proteins to amylose resin in the presence of 6 µg of untagged MCM or PCNA proteins. Lane 1, molecular weight marker; lanes 2 and 3, MCM alone; lanes 4 and 5, MCM and MBP-Cdc6-1; lanes 6 and 7, MCM and MBP-Cdc6-2; lanes 8 and 9, PCNA alone; lanes 10 and 11, PCNA and MBP-Cdc6-1; lanes 12 and 13, PCNA and MBP-Cdc6-2. Lanes 2, 4, 6, 8 and 10 contain 10% of the reaction mixture and are marked by ‘L’. Lanes 3, 5, 7, 9 and 11 contain the proteins eluted from the amylose resin and are marked by ‘P’.
Figure 4
Figure 4
Cdc6–MCM interaction is required for the inhibition of MCM translocation along DNA. MCM helicase translocation along ssDNA (A–C) and dsDNA (D–F) was assayed as described in Materials and Methods in the presence of 0.3 pmol MCM (as monomer) and increasing amounts of Cdc6-1 and -2 proteins and their derivatives. (A, B, D and F) show representative gels. Lane 1, substrate only; lane 2, boiled substrate; lane 3, no Cdc6; lanes 4, 7, 10, 13 and 16, 0.3 pmol of Cdc6 protein as monomer; lanes 5, 8, 11, 14 and 17, 1.2 pmol of Cdc6 protein as monomer; lanes 6, 9, 12, 15 and 18, 4.8 pmol of Cdc6 protein as monomer. 32P-labeled strands are marked by an asterisk. (C and F) summarize the percent inhibition of MCM translocation (C) and duplex translocation (F) observed in the presence of the various Cdc6 proteins. Error bars represent the standard deviation calculated from 3 experiments. Top: Schematic representation of the Cdc6-1 and -2 proteins used in the study.
Figure 5
Figure 5
An intact WH domain of Cdc6 is needed for DNA binding. Filter binding assays were performed as described in Materials and Methods using 32P-labeled single-stranded or dsDNA oligonucleotides in the presence of 0.1, 0.3, 0.9 and 2.7 pmol of proteins (as monomer). The averages with standard deviations of three experiments are shown. (A) dsDNA binding of Cdc6-1 proteins; (B) ssDNA binding of Cdc6-1 proteins; (C) dsDNA binding of Cdc6-2 proteins; (D) ssDNA binding of Cdc6-2 proteins.
Figure 6
Figure 6
Cdc6 autophosphorylation is regulated by MCM binding. Cdc6 autophosphorylation reactions were performed as described in Materials and Methods in a reaction mixture (15 µl) containing 10 pmol of Cdc6 protein and 3.3 pmol of [γ-32P]ATP in the absence (lanes 3, 5, 7, 9, 11 and 13) or in the presence (lanes 4, 6, 8, 10, 12 and 14) of 20 pmol MCM. The autophosphorylation reactions were carried out for 20 min at 65°C. Following incubation, the proteins were separated by 10% SDS–PAGE and visualized by Coomassie blue staining (A) and autoradiography (B). Lane 1, molecular mass (kDa); lane 2, MCM alone; lanes 3 and 4, Cdc6-1 full-length protein; lanes 5 and 6, Cdc6-1 truncated proteins; lane 7 and 8, Cdc6-1 full-length protein with mutant WH domain; lanes 9 and 10, Cdc6-2 full-length; lanes 11–12, Cdc6-2 truncated proteins; lane 7 and 8, Cdc6-2 full-length protein with a mutated WH domain. A representative gel is shown.
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