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Journal of Bacteriology, June 2006, p. 4577-4580, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.00168-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DNA Binding by the Methanothermobacter thermautotrophicus Cdc6 Protein Is Inhibited by the Minichromosome Maintenance Helicase

Rajesh Kasiviswanathan, Jae-Ho Shin, and Zvi Kelman^*

University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, Maryland 20850

Received 31 January 2006/ Accepted 5 April 2006

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The Cdc6 proteins from the archaeon Methanothermobacter thermautotrophicus were previously shown to bind double-stranded DNA. It is shown here that the proteins also bind single-stranded DNA. Using minichromosome maintenance (MCM) helicase mutant proteins unable to bind DNA, it was found that the interaction of MCM with Cdc6 inhibits the DNA binding activity of Cdc6.

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The initiation of DNA replication requires the recognition of the origin of replication by specific proteins. Among those are the Escherichia coli DnaA protein and the eukaryotic origin recognition complex (ORC). The archaeon Methanothermobacter thermautotrophicus contains two homologues of ORC, referred to as Cdc6-1 and Cdc6-2. It is thought that these archaeal proteins function in origin recognition and helicase loading. The organism also contains a single homologue of the minichromosome maintenance (MCM) helicase, thought to function as the replicative helicase (9).

Studies have shown that the two Cdc6 proteins interact with double-stranded DNA (dsDNA) via the winged-helix (WH) domain (6, 7). It was also shown that the Cdc6 proteins bind origin-derived duplex DNA containing inverted repeats with greater affinity than that for random DNA sequences (2). Both specific and nonspecific DNA binding is mediated via the WH domains of the proteins (2, 6, 7). MCM interaction with DNA is sequence independent, as expected from a replicative helicase that unwinds chromosomal DNA. Two structural features in MCM were shown to be involved in DNA binding. A zinc finger motif was found to participate in single-stranded DNA (ssDNA) binding (14), and a role for the ß-finger motif in dsDNA binding has also been reported (4).

The mechanism of origin recognition and helicase loading is currently unknown. In a step towards elucidating the mechanism of helicase loading, it was shown that the interactions between Cdc6 and MCM inhibit MCM helicase activity (7, 16). This observation is reminiscent of the observation made for bacteria in which the binding of the helicase loader, DnaC, to the helicase, DnaB, inhibits helicase activity.

In this study, the effect of Cdc6-MCM interactions on DNA binding by Cdc6 was determined.

M. thermautotrophicus Cdc6-1 and Cdc6-2 proteins bind differently to DNA. Previous studies, using a 282-bp DNA fragment derived from the M. thermautotrophicus putative origin of replication (10), demonstrated that both the Cdc6-1 and Cdc6-2 proteins bind to duplex origin DNA (2). A filter binding assay (2, 7) was used to determine whether the proteins can bind oligonucleotides containing three tandem repeats found within the origin (5'-TTTACACTTGAAAGGGTTTACACTTGAAAGGGTTTACACTTGAAA-3') or random sequences (5'-TACATATGTACATGGGTACATATGTACATGGGTACATATGTACAT-3') which were generated by maintaining the same base composition as that of the origin sequences but scrambling the order. As shown in Fig. 1, and similar to the work by Capaldi and Berger (2), both proteins bind to dsDNA containing the origin-derived inverted repeats (Fig. 1A and B). Cdc6-2 bound efficiently to both specific and random DNA (Fig. 1B). Cdc6-1 protein, on the other hand, showed some preference for origin-specific double-stranded sequences (Fig. 1A).

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FIG. 1. Cdc6-1, but not Cdc6-2, protein exhibits preferential binding to origin DNA. DNA binding analyses of Cdc6-1 (A and C, filled symbols) and Cdc6-2 (B and D, filled symbols) and Cdc6-1 WH mutant (A and C, open symbols) and Cdc6-2 WH mutant (B and D, open symbols) proteins were performed as previously described (7) using filter binding assays in the presence of 2.5 nM 32P-labeled origin-specific (circles) or random (triangles) DNA sequences in the presence of 15, 30, 45, 90, 150, and 225 nM concentrations of Cdc6 protein (as a monomer). (A and B) dsDNA; (C and D), ssDNA. The average result of three experiments is shown with standard deviations.

Next, the ability of the proteins to bind ssDNA was determined using origin-specific and random DNA. While Cdc6-2 binds both ssDNA substrates efficiently (Fig. 1D), no ssDNA binding could be detected with Cdc6-1 (Fig. 1C).

It was shown that the WH motif at the C terminus of the Cdc6 proteins is required for dsDNA binding (2, 6). The role of the domain in ssDNA binding was evaluated using proteins with mutations in the WH domain. The mutation in Cdc6-1 was identical to that previously reported to abolish DNA binding, replacing Arg₃₃₄ and Arg₃₃₅ with Ala (2). In Cdc6-2, there is only a single Arg residue (Arg₃₃₇) in a similar but not identical location, and it was also replaced by Ala. When the mutant proteins were used, it was found that while the mutation in Cdc6-1 completely abolished dsDNA binding (Fig. 1A), the mutation in Cdc6-2 reduced dsDNA binding by about 50% (Fig. 1B) and ssDNA binding by about 90% (Fig. 1D).

The data presented in Fig. 1 suggest that the WH domains play a major role in DNA binding by Cdc6. However, neither the intact WH domain nor a truncated enzyme in which the WH domain was deleted binds DNA (reference 7 and data not shown), suggesting that a full-length Cdc6 protein is needed for DNA binding. To test whether a chimeric protein containing the N-terminal catalytic domain from one Cdc6 and the C-terminal WH domain from the other (Cdc6-N1C2 and Cdc6-N2C1) (Fig. 2) could bind DNA, the proteins were constructed. As shown in Fig. 2A and B, neither chimera could interact with DNA. These results suggest not only that both the WH and catalytic domains are needed for DNA binding but also that only a WH and a catalytic domain from the same protein can bind DNA.

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FIG. 2. Cdc6 chimeric proteins cannot bind DNA. (Top) Schematic representation of the Cdc6 chimeric proteins. DNA binding by Cdc6-1 (A and B, open circles), Cdc6-2 (A and B, open triangles), and the chimeras, namely Cdc6-N1C2 (A and B, closed triangles) and Cdc6-N2C1 (A and B, closed circles), were measured using filter binding assays as previously described (7) in the presence of 2.5 nM 32P-labeled origin-specific ssDNA (A) or dsDNA (B) in the presence of 15, 30, 45, 90, 150, and 225 nM concentrations of protein (as a monomer). The average result of two experiments is shown. (C) The effect of full-length and chimeric Cdc6 proteins on the helicase activity of MCM was determined as previously described (16) in the presence of 13.3 nM MCM (as a monomer) and 26.7 nM (lanes 4, 7, 10, and 13), 80 nM (lanes 5, 8, 11, and 14), and 240 nM (lanes 6, 9, 12, and 15) concentrations of the Cdc6 proteins (as monomers). Percent inhibition of helicase activity in comparison to the reaction without Cdc6 (lane 3) is indicated above the lane numbers. S, substrate; P, product.

It was previously shown that when the Cdc6 proteins interact with MCM, they inhibit its helicase activity (7, 16). Thus, the inhibition of helicase activity could suggest that the chimeras are properly folded. Both chimeric proteins inhibit MCM helicase activity (Fig. 2C).

The ß-finger of the M. thermautotrophicus MCM protein is required for both ssDNA and dsDNA binding. It was previously shown that two motifs located in the N-terminal part of MCM play a role in DNA binding. A zinc finger motif is needed for ssDNA binding (14), while a ß-finger is required for duplex interactions by the N-terminal part of the molecule (4). The roles of the zinc finger in dsDNA binding and that of the ß-finger in ssDNA binding are not yet known. Thus, a zinc finger mutant protein in which Cys₁₅₈, which is a part of the zinc finger, was replaced by Ser (14) and a ß-finger mutant protein in which R₂₂₇ and K₂₂₉ were replaced by Ala (4) were examined for the ability to bind ssDNA and dsDNA. As shown in Fig. 3, both mutant proteins are impaired for DNA binding. The zinc finger mutant retained the ability to interact with DNA, although binding was less efficient than that of the wild-type enzyme. The mutations in the ß-finger motif, on the other hand, completely abolished the ability of MCM to interact with DNA (Fig. 3).

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FIG. 3. The ß-finger of MCM is essential for ssDNA and dsDNA binding. DNA binding by MCM and MCM mutant proteins was measured using filter binding assays as previously described (7) in the presence of 2.5 nM 32P-labeled random ssDNA (open symbols) or dsDNA (closed symbols) in the presence of 15, 30, 45, 90, 150, and 225 nM concentrations of MCM (as a monomer). Circles, wild-type enzyme; squares, zinc finger mutant; triangles, ß-finger mutant. The average result of three experiments is shown with standard deviations.

MCM-Cdc6 interactions inhibit Cdc6 DNA binding. Cdc6-1 and Cdc6-2 were shown to interact with MCM (7, 16), and these interactions inhibit MCM helicase activity (7). In order to gain insight into the possible mechanism of helicase loading, the effect of MCM interaction with Cdc6 on the DNA binding activity of Cdc6-1 and Cdc6-2 was determined. For this study, the ß-finger mutant of MCM was used, as it is devoid of DNA binding activity (Fig. 3) and thus will not create background in the experiment, yet it retains the ability to interact with Cdc6-1 and Cdc6-2 (7). The presence of MCM substantially reduced ssDNA and dsDNA binding by Cdc6-1 and Cdc6-2 (Fig. 4). As Cdc6-1 did not bind ssDNA (Fig. 1C), MCM has no effect on this reaction and thus is not shown in Fig. 4B. These results show that MCM-Cdc6 interactions affect the DNA binding activity of Cdc6.

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FIG. 4. MCM inhibits DNA binding by Cdc6. DNA binding assays were performed as previously described (7) using 2.5 nM 32P-labeled origin-specific dsDNA (A) or ssDNA (B) with 15, 30, 45, 90, and 150 nM concentrations of Cdc6-1 (circle) and Cdc6-2 (triangle) in the presence (filled symbols) or absence (open symbols) of a 300 nM concentration of the ß-finger mutant of MCM. The average result of three experiments is shown with standard deviations.

The role of MCM in the regulation of DNA binding by Cdc6 is not yet known. However, MCM may participate in a switch mechanism. Upon MCM loading at the origin by Cdc6, MCM will take its place on the DNA. Cdc6, however, may still bind to the MCM, as it was shown that the MCM helicase activity is inhibited under these conditions. Other factors or processes (e.g., autophosphorylation) may remove the Cdc6 protein to initiate DNA unwinding.

Cdc6-1 and Cdc6-2 may have different roles during the initiation of DNA replication. Many archaeal genomes contain two Cdc6 homologues. It was proposed that one protein is the functional homologue of the eukaryotic Cdc6 and that the other is the functional homologue of ORC, involved in origin recognition (8). In addition, it was shown that most archaeal origins identified to date are located in the vicinity of a gene encoding Cdc6 (11, 12, 15), suggesting that the product of this gene is the origin binding protein (13).

In M. thermautotrophicus, the gene encoding Cdc6-1 is located immediately upstream of the origin of replication (10), and thus it is the prime candidate to function as the origin recognition factor. The data presented here together with those from past studies (2) support this hypothesis, as Cdc6-1 shows preferential binding to inverted repeats found within the origin region rather than to random DNA sequences.

The archaeal helicase loader has not yet been identified. However, the data presented here, other observations, and the similarities to the bacterial and eukaryal systems suggest that Cdc6-2 may play a role in helicase assembly at the origin. The protein has amino acid sequence similarity to the eukaryotic Cdc6 (5), which participates in MCM assembly at the eukaryotic origin (1). In bacteria, when the helicase loader, DnaC, associates with the replicative helicase, DnaB, it inhibits its helicase activity. Inhibition is relieved upon helicase loading at the origin (3). Similar observations were made with the archaeal Cdc6 proteins, which also inhibit MCM helicase activity (16). In M. thermautotrophicus, it was shown that Cdc6-2 inhibits helicase activity more than Cdc6-1 (7, 16). In addition, as shown in Fig. 1, Cdc6-2 does not have any clear DNA binding preference for origin sequence. Thus, taken together, these observations may support the idea that in M. thermautotrophicus, Cdc6-2 protein participates in helicase loading.

The results presented here together with observations previously made suggest that in M. thermautotrophicus, Cdc6-1 is the functional homologue of ORC and that Cdc6-2 participates in helicase loading at the origin. This may be supported by the observation that the archaeal Cdc6 proteins can be divided into two distinct groups based on sequence conservation of the motifs involved in DNA recognition (5, 17). Since many archaeal species contain two Cdc6 homologues, the proteins may have separate functions in other species as well.

 
We thank Lori Kelman and the members of the laboratory for their comments on the manuscript.

This work was supported by a research scholar grant from the American Cancer Society (RSG-04-050-01-GMC).

 
* Corresponding author. Mailing address: University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, 9600 Gudelsky Drive, Rockville, MD 20850. Phone: (240) 314-6294. Fax: (240) 314-6255. E-mail: kelman{at}umbi.umd.edu.

Present address: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale Street, Memphis, TN 38105.

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Journal of Bacteriology, June 2006, p. 4577-4580, Vol. 188, No. 12
0021-9193/06/$08.00+0     doi:10.1128/JB.00168-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kasiviswanathan, R. Articles by Kelman, Z. Search for Related Content PubMed PubMed Citation Articles by Kasiviswanathan, R. Articles by Kelman, Z.