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J. Biol. Chem., Vol. 278, Issue 49, 49053-49062, December 5, 2003
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From the
University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, Rockville, Maryland 20850 and Program in Molecular Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, August 5, 2003 , and in revised form, September 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The minichromosome maintenance (MCM) complex is believed to function as the replicative helicase of Eukarya and Archaea, suggesting that it is the functional homologue of the bacterial DnaB protein (12–14). In Eukarya, MCM is a family of six proteins (Mcm2–7) with highly conserved amino acid sequences between the six different polypeptides (13). All MCM proteins are essential for cell viability and have been identified in all organisms studied (13). In addition to forming a heterohexamer, in vivo and in vitro studies have revealed the presence of additional MCM complexes composed of different combinations of the MCM proteins (12, 15, 16). Biochemical studies with the various complexes in yeast and mammals have shown that a dimeric complex of the Mcm4,6,7 heterotrimer contains 3' 
5' DNA helicase, ssDNA binding, and DNA-dependent ATPase activities, whereas its interactions with either Mcm2 or Mcm3,5 inhibit the helicase activity (12, 15, 17). All six proteins, however, were shown to be essential for replication fork movement (18), and mutational analyses have shown that physical interactions between specific members of the two subgroups (Mcm4,6,7 and Mcm2,3,5) are required for efficient ATPase activity (19, 20).
In Archaea, a single MCM protein homologue has been identified in all organisms studied (21, 22). Biochemical studies with the MCM proteins from Methanothermobacter thermautotrophicus (mt) (4, 23–25) and Sulfolobus solfataricus (26) revealed that these enzymes possess biochemical properties similar to those of the eukaryotic Mcm4,6,7 complex including 3' 5' helicase, ssDNA binding, and DNA-dependent ATPase activities. The mtMCM has been shown to exist as a double hexamer in solution (4, 23).
Several in vivo observations made with the eukaryotic MCM complexes suggest that these proteins may bind dsDNA in addition to ssDNA. It was shown that the MCM proteins preferentially bind to unreplicated DNA (27, 28) and that the complex is distributed over large regions of DNA surrounding origins (29). The majority of the MCM proteins are not localized at replication forks where DNA synthesis takes place and where ssDNA regions are available (27, 30). These and other observations prompted a proposal that the MCM complex may translocate along duplex DNA following loading at the origin (31). To date, however, dsDNA translocation by MCM has not been reported.
Support for the possible translocation of the MCM complex along duplex DNA comes from studies with the bacterial DnaB and phage-T7 gp4 helicases. Using Thermus aquaticus and Escherichia coli DnaB proteins (32, 33) and phage-T7 gp4 helicase (33), it was demonstrated that in the presence of dsDNA containing a 5'-ssDNA tail, the helicases encircle both strands of the duplex and translocate along the duplex. Their transit along dsDNA, similar to the unwinding reactions, requires NTP hydrolysis. The diameter of the central channel of these helicases, including the mtMCM complex, varies between 
20 and 45 Å, large enough to accommodate the passage of dsDNA (1, 34–36).
In this study we examined the ability of the spMcm4,6,7 complex and the mtMCM protein to encircle and translocate along duplex DNA. The data presented here demonstrate that both helicases can translocate along dsDNA. The study also shows that during its movement along DNA the archaeal protein, but not the eukaryal complex, can displace streptavidin linked to biotinylated oligonucleotides present in either single-stranded or duplex regions.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-32P]ATP were obtained from Amersham Biosciences, and oligonucleotides and biotinylated oligonucleotides were synthesized by the Center for Advanced Research in Biotechnology DNA synthesis facilities. Biotin was obtained from Sigma, streptavidin was from Rockland Inc., and Biotin-dT (5'-dimethoxytrityloxy-5-[N-(((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2'-deoxyuridine-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) was obtained from Glen Research. The proteins, mtMCM, mtMCM K325A mutant, and the spMcm2–7 and spMcm4,6,7 complexes were purified as described previously (4, 15).
Methods
Preparation of Substrates for Helicase Assays—Oligonucleotides used for the preparation of the different helicase substrates are listed in Table I. The helicase substrates were made by labeling one or two of the oligonucleotides (as indicated in the figure legends) at the 5' end using [-32P]ATP and T4 polynucleotide kinase. Labeling reactions were stopped by adding EDTA to a final concentration of 25 mM. The labeled oligonucleotide(s) were hybridized to the other oligonucleotides (usually at a 1:1 molar ratio) in a buffer containing 40 mM Hepes-NaOH (pH 7.5) and 50 mM NaCl by heating to 100 °C for 3 min followed by slow cooling to 25 °C. Unincorporated [-32P]ATP and unannealed oligonucleotides were removed using the following procedure. After hybridization, a 6x loading buffer (0.1% xylene cyanol, 0.1% bromophenol blue, and 50% glycerol) was added to a final concentration of 1x, and the mixture was electrophoresed through an 8% native polyacrylamide gel for 1 h at 100 V in 0.5x TBE (45 mM Tris, 4.5 mM boric acid, 0.5 mM EDTA). The desired substrates were located by autoradiography, and the products were excised from the gel, which was sliced into small pieces and incubated at 37 °C for 2 h in 3 volumes of an elution buffer containing 0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH 8.0). After centrifugation, the supernatant was collected, and the insoluble material was extracted once more with elution buffer. Following centrifugation, both supernatant fractions were combined, and DNA substrates were then ethanol precipitated and dissolved in TE (10 mM Tris-HCl (pH 7.5), 1 mM EDTA). For assays involving biotin-streptavidin complexes, the biotinylated DNA substrates were incubated with 50 nM streptavidin for 10 min at 30 °C prior to use.
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DNA helicase activity of the archaeal enzyme was measured in reaction mixtures (15 µl) containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 2 mM dithiothreitol, 100 µg/ml bovine serum albumin, 5 mM ATP, 10 fmol of 32P-labeled DNA substrate (3,000 cpm/fmol), and the amount of MCM protein as indicated in the figure legends. Mixtures were incubated at 60 °C for 1 h (except when indicated otherwise in the figure legends) and analyzed as described for the spMcm4,6,7. All helicase experiments described below (see Figs. 1, 2, and 4) were repeated three-five times with almost identical results. Representative gels are shown in each figure.
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-32P]ATP and T4 polynucleotide kinase at their 5' end and purified as described above for the preparation of helicase substrates. Ten fmol of ss or ds oligonucleotides (as indicated in figure legends) were incubated with 50 nM streptavidin in the helicase reaction mixture (15 µl) at 30 °C for 10 min, followed by the addition of 50 µM free biotin (to trap and sequester streptavidin if released during the helicase reaction) and spMcm4,6,7 or mtMCM (as indicated in the figure legends). After incubation for 1 h at 30 °C (spMCM4,6,7) or 60 °C (mtMCM), 5 µl of 5x loading buffer was added to each sample, followed by electrophoretic analysis on a native 8% polyacrylamide gel in 0.5x TBE. The gels were analyzed using phosphorimaging. All experiments described below in Fig. 3 were repeated five times with almost identical results. Representative gels are shown in the figure.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5' polarity of the enzyme, was used to design various substrates to examine whether the spMcm4,6,7 complex translocates along dsDNA. Oligonucleotides were constructed and hybridized together to produce the desired ss and dsDNA regions as shown below. All DNA substrates used in these studies contained duplex regions of 25 bp in length. The substrate shown in Fig. 1A, consisting of three oligonucleotides hybridized together, contained two 25-bp dsDNA regions separated by a nick on the bottom strand, whereas the top strand was hybridized contiguously to both duplexes. The left dsDNA region contained a 24-nt 3'-ssDNA overhang whereas the duplex region on the right had a 24-nt 5'-ssDNA overhang. The two bottom strands were labeled at their 5' ends.
Because spMcm4,6,7, which moves in the 3' 5' direction, cannot displace a 25-bp duplex in the absence of a fork structure (37), an unwinding reaction carried out with the substrate shown in Fig. 1A could yield several products. (i) The protein complex binds to the 3'-ssDNA overhang region and translocates until it reaches the duplex region and stops, resulting in no strand displacement. (ii) Upon encountering the left duplex, the protein translocates along this duplex region by encircling both strands within its central channel and continues moving through the duplex until it reaches the right duplex, which it then displaces. (iii) The complex assembles at the fork-like structure of the right duplex and displaces the duplex region. In this scenario, the helicase may dimerize so that one hexamer encircles the duplex whereas the other hexamer encircles the ssDNA region, as demonstrated previously (37) for forked DNA structures. In the second and third alternatives the right duplex region is displaced, whereas the left duplex region remains associated with the top strand.
Experimentally, in the presence of increasing levels of the spMcm4,6,7 complex, only the right duplex was displaced with the accumulation of the product containing the top strand hybridized to the left duplex region (Fig. 1A). However, no product containing only the right duplex region hybridized to the top strand was detected (if formed it would be located where indicated on the figure). Similar results were observed with a substrate in which the 5'-ssDNA overhang in the right duplex region was 36 bp long (data not shown). These results can be explained by either the second or third possibility described above. To distinguish between these two, the substrate shown in Fig. 1B was constructed. This substrate is similar to that used in Fig. 1A, but the 3'-ssDNA overhang in front of the left duplex region was removed, leaving only the 5'-ssDNA overhang. As shown, no displacement of either duplex region was detected (Fig. 1B). To determine whether a longer duplex region was needed to assemble the helicase on DNA, a substrate in which the left duplex region was extended to 50 bp was analyzed. Similar to the results shown in Fig. 1B, no displacement of either duplex was observed (data not shown). These results demonstrate that the spMcm4,6,7 complex does not assemble at the fork and that the enzyme does not translocate from dsDNA ends. The results also suggest that the enzyme required a 3'-overhang region to initiate movement along dsDNA.
To further test the need for the 3'-ssDNA overhang region, the substrate shown in Fig. 1C was made. This substrate is similar to that used in Fig. 1A but contained a 5'-ssDNA overhang region instead of a 3'-ssDNA overhang. No helicase activity was observed with this substrate, substantiating the conclusion that a 3'-ssDNA overhang is required to support the displacement of the right duplex.
The results presented in Fig. 1, A–C suggest that the spMcm4,6,7 complex assembled at the 3'-ssDNA overhang and then translocated along the left duplex region until it reached the right duplex with its fork-like structure. To test this hypothesis, DNA substrates containing biotin-streptavidin blocks were used (Fig. 1, D–I). First, the ability of streptavidin to block the movement of the spMcm4,6,7 complex along ssDNA regions was examined, and the results are shown in Fig. 1, D and E. This substrate contained a single 25-bp duplex and both 3' and 5'-ssDNA overhang regions. A biotinylated nucleotide was incorporated at the 3'-overhang region (the strand on which the helicase binds and translocates). Upon binding streptavidin, the biotin could block the movement of the helicase. In this DNA substrate, the oligonucleotide strand devoid of biotin was labeled. As shown in Fig. 1D, the substrate containing only the biotinylated nucleotide did not block helicase activity. In contrast, when streptavidin was bound to the biotinylated oligonucleotide, helicase activity was not detected (Fig. 1E). When the biotin-streptavidin was placed on the complementary strand (the strand on which the helicase does not bind and move), the helicase displaced this strand (Fig. 1F). Thus, the presence of a large protein complex on the translocating strand prevents helicase activity, presumably by blocking helicase movement.
A similar approach was used to determine whether the results shown in Fig. 1A were because of translocation of the helicase through the left duplex region. For this purpose, the substrate used in Fig. 1A was modified so that the bottom strand of the left duplex contained the biotinylated nucleotide. If dsDNA translocation were required then the presence of the streptavidin-biotin complex could block the movement of the spMcm4,6,7 complex. As shown in Fig. 1G, the presence of biotin alone did not affect the displacement of the right duplex region whereas the streptavidin-biotin complex prevented its displacement (Fig. 1H). As expected, the presence of the biotin-streptavidin complex on the bottom strand of the right duplex region had no effect on its displacement (Fig. 1I).
Taken together, the findings described in Fig. 1, A–I suggest that the spMcm4,6,7 complex translocates along dsDNA, provided a 3'-ssDNA overhang region is present to initiate the translocation. These results are similar to those observed with the T. aquaticus DnaB helicase (32) with the exception that because of the 5' 3' directionality of movement of DnaB, a 5'-ssDNA overhang region is required rather than a 3'-ssDNA overhang. As observed with the DnaB helicase, ATP hydrolysis was essential for all displacement reactions catalyzed by the eukaryotic helicase. The omission of ATP or the use of ATPS in place of ATP did not support helicase activity.
As an additional control, a double forked duplex substrate was constructed (Fig. 1J). Because the spMcm4,6,7 complex displaces a 25-bp duplex region in the presence of a fork structure (37), we expected that the left duplex region would be displaced first followed by the unwinding of the right duplex. As shown in Fig. 1J, this was indeed the case. This is illustrated by the displacement of the 76-mer oligonucleotide (left duplex) at low enzyme concentrations, prior to the appearance of the single stranded 61-mer (right duplex) (compare lanes 5 and 3 in Fig. 1J). Thus, when the helicase complex is assembled at a fork structure, dsDNA translocation is not observed.
The eukaryotic MCM proteins form several complexes including the heterohexamer Mcm2–7 (15). To date, in vitro studies with the Mcm2–7 complex from different organisms have failed to detect helicase activity with DNA substrates containing 3'-ssDNA overhang regions or forked structures (16). The possibility that the heterohexameric complex translocated along duplex DNA that, in turn, activated the helicase activity of spMcm4,6,7 was examined. However, the spMcm2–7 complex failed to displace the duplex regions of substrates shown in Fig. 1, A and B (data not shown). These findings do not exclude the possibility that at the origin, during the initiation phase of replication, such an activity could be observed. Thus, conditions that initiate the putative helicase activity of the Mcm2–7 complex remain unknown.
The Archaeal MCM Is Capable of Translocating along Duplex DNA—Translocation of the archaeal MCM along dsDNA was also examined using the approach described above. It was shown previously (4, 23, 24) that the mtMCM can displace long duplex regions even in the absence of forked structures, although the enzyme acts more efficiently with forked structures (38). Thus, as expected, when the substrate described in Fig. 1A was used in the helicase assay, both the left and right duplex regions were displaced by the mtMCM complex (Fig. 2A). The results could be explained by the assembly of the mtMCM helicase on the 3'-ssDNA overhang followed by its translocation along the top strand in the 3' 5' direction. This would result in the displacement of the left duplex region followed by the displacement of the right duplex. Alternatively, helicase molecules could assemble simultaneously on the 3'-ssDNA overhang region and the fork-like structure formed by the right duplex. In this case, two identical helicase complexes could act independently to displace the two duplexes. The results presented in Fig. 2A revealed the formation of an intermediate containing the left duplex region hydrogen bonded to the top strand at low enzyme concentrations. However, no product containing only the right duplex region hybridized to the top strand was detected (if formed it would be located where indicated on the figure). Similar results were obtained when the rate of unwinding was examined with this substrate (Fig. 2B). These results exclude the first possibility involving the sequential displacement of the 25-mer followed by the displacement of the 49-mer oligonucleotide with its fork-like structure. It is possible that mtMCM proteins assembled independently in front of both duplex regions and that the complex associated with the 49-mer fork-like structure was a more active helicase, in keeping with the finding that its helicase activity is enhanced by forked DNA structures (38). Alternatively, upon assembly at the 3'-ssDNA overhang region some of the complex, instead of encircling the ssDNA and displacing the duplex region ahead, encircled and translocated along the duplex, as observed with the spMcm4,6,7 complex (Fig. 1A), until it reached and subsequently displaced the second fork-like duplex structure.
In Fig. 2C the requirement for a 3'-ssDNA overhang region was analyzed using a substrate similar to that described in Fig. 2A but without the 3'-ssDNA overhang region. In contrast to the spMcm, the mtMCM displaced the right duplex region in the absence of a 3'-ssDNA overhang region. Unlike the results shown in Fig. 2A, however, the left duplex region remained associated with the top strand suggesting that a 3'-ssDNA overhang is required for its displacement. Similar results were obtained with a longer (50 bp) left duplex. The results shown in Fig. 2C suggest that the mtMCM helicase translocated along the duplex or was assembled at the fork in front of the right duplex region. Support for these possibilities comes from analyzing the products formed with the substrate described in Fig. 2D. This substrate contained two 5'-ssDNA overhang regions but no 3'-ssDNA overhang region. As shown, mtMCM displaced only the right duplex region, suggesting that the helicase assembled directly on the duplex portion. It should be noted that assembly of the mtMCM on the two ssDNA overhang regions would not lead to displacement of the duplex. Upon binding to these 5'-ssDNA overhang regions, the enzyme would translocate away from the duplex because of the 3' 5' polarity of movement.
In contrast to the differences noted with the DNA substrates described in Fig. 2, A–D, both spMcm and mtMCM complexes acted identically with the double forked duplex substrate shown in Fig. 2E. Both enzymes displaced the left duplex first followed by unwinding of the right duplex (see Fig. 1J and Fig. 2E). Thus, in the presence of 3'- and 5'-ssDNA tailed duplex DNAs, both enzymes bind to and translocate along the 3'-ssDNA tailed region toward the 5' end and displace duplex regions in their path. Like the spMcm4/6/7 complex, the unwinding reactions catalyzed by the mtMCM complex required ATP hydrolysis. No duplex unwinding was observed in the absence of ATP or with non-hydrolyzable ATP derivatives (AMP-PNP or ATPS).
To examine whether the mtMCM translocated along dsDNA, dsDNAs containing biotin-streptavidin blocks were examined in experiments similar to those carried out with the eukaryotic enzyme (Fig. 2, F–H). Unlike the eukaryotic enzyme, the biotin-streptavidin complex did not block mtMCM translocation and helicase activities, regardless of whether the streptavidin was linked to the ss (Fig. 2G) or dsDNA regions (Fig. 2I). The possibility that mtMCM displaces streptavidin from biotinylated oligonucleotides was considered, because previous studies demonstrated that phage-T4 gp41 helicase could displace streptavidin from biotinylated oligonucleotides (39). For this purpose streptavidin-biotin complexes bound to ss or ds oligonucleotides were incubated with increasing levels of helicase in the presence of a large excess of biotin, which served to bind streptavidin upon its displacement from the DNA. As shown in Fig. 3, A and B, spMcm4,6,7 did not displace streptavidin from either ss or ds biotinylated DNA. With both substrates a significant portion of the oligonucleotide did not bind streptavidin for reasons presently unclear. mtMCM, on the other hand, displaced streptavidin from the biotinylated DNAs (Fig. 3, C and D). ATP was required for this reaction (compare lanes 7 and 6) and a K325A mutant of mtMCM, shown previously (23–25) to be devoid of helicase activity, did not displace streptavidin in the presence of ATP (Fig. 3C, compare lanes 8 and 6). Taken together, these data show that translocation of mtMCM along ss and dsDNAs can lead to streptavidin displacement from biotinylated oligonucleotides. This protein displacement reaction explains why the streptavidin-biotin complex did not block the movement of mtMCM along ss and dsDNAs (Fig. 2, G and I).
MCM Stops DNA Unwinding When Encountering DNA Nicks—After establishing that both the archaeal and eukaryal MCM helicases can move along duplex DNA, a possible role for this movement was investigated. It was hypothesized previously (32) that helicase translocation along dsDNA may play a role in preventing DNA unwinding when the helicase encounters nicks in the DNA and switches from DNA unwinding to dsDNA movement. Thus, the effect of DNA nicks in either the translocating strand (the leading strand at the replication fork) or in the complementary strand (the lagging strand at the replication fork) on helicase activity were examined. As shown in Fig. 4, C and D, the spMCM4,6,7 complex stopped displacing the duplex when it encountered a nick on the DNA regardless of whether the nick was on the translocating (Fig. 4D) or complementary strand (Fig. 4C). As spMCM4,6,7 requires a fork structure to initiate helicase activity (Fig. 1), no displacement was detected with substrates containing only 3'-overhanging DNA regions (Fig. 4, A and B). These results suggest that when the nick is present on the translocating strand the helicase falls off the template upon encountering the nick (Fig. 4I). When the nick is on the lagging strand the helicase may switch from DNA unwinding to dsDNA movement at the nick, resulting in no further DNA displacement (Fig. 4J).
The results with the archaeal enzyme were somewhat different, because this helicase, in contrast to the eukaryal complex, displaced duplex substrates with only a 3'-overhang region (Fig. 2A) and could load onto recessed 3'-substrates and then translocate through the duplex (Fig. 2D). As shown in Fig. 4E, the mtMCM complex displaced the nicked lagging strand (non-translocating strand). When the nick was on the translocating strand, the labeled 80-nt region was displaced whereas the 40-nt duplex downstream of the nick was not (Fig. 4F). Possibly the enzyme dissociated from the substrate or moved through the duplex upon encountering the nick. Identical results were obtained with nicked forked DNA substrates. Displacement of the nicked lagging strand was observed (Fig. 4G) whereas DNA containing a nick on the leading strand only supported displacement of the duplex region upstream of the nick (Fig. 4H). Thus, the mechanism of action of the two helicases differed selectively in their activity with substrates containing a nick on the lagging strand as summarized in Fig. 4J.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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5' DNA helicase, ssDNA binding, and DNA-dependent ATPase activities. In this report, we have demonstrated that both complexes are capable of binding to a 3'-ssDNA tailed region of a forked duplex DNA and translocating on this single-stranded region in the 3' 5' direction. Based on current models, it is likely that the bound 3'-ssDNA region is placed inside the central channel of these hexameric complexes with the 5'-ssDNA tail positioned outside the channel (1). Further translocation through the duplex results in unwinding of the dsDNA. A different mechanism is evident with duplex substrates containing only a 3'-ssDNA tail. With such substrates, the ring-shaped MCM helicases actively move along the duplex with both strands most likely positioned within the central channel and exhibit helicase activity only in the presence of a downstream forked duplex structure. This mode of movement was evident with the spMcm complex only on DNA substrates lacking a 5'-ssDNA tail. Thus, with the spMcm helicase, the presence or absence of a forked structure (and specifically the 5'-ssDNA tail) determines whether a single strand (resulting in unwinding) or the duplex is placed within the central channel. The movement and helicase activity of the spMcm complex was blocked by the presence of a streptavidin-biotin complex on the 3'-strand (to which the helicase binds) but not by a streptavidin-biotin complex on the complementary strand. Movement along dsDNA, however, was blocked by the presence of a streptavidin-biotin complex on either strand of the duplex (data not shown).
Though the translocation properties of the mtMCM and spMcm complex are similar, specific differences were noted. These differences included the following. (i) The mtMCM displaced duplex regions with a 3'-ssDNA overhang but without a forked structure, whereas the eukaryotic enzyme did not. (ii) In contrast to the spMcm complex, the mtMCM enzyme translocated along flush ended dsDNA and recessed 3' ends to displace a downstream forked duplex. (iii) Movement of the mtMCM was unimpeded by a streptavidin-biotin complex regardless of its location on DNA. Because the diameter of streptavidin (50Å (40)) is larger than the central channel of the mtMCM complex (23–47Å (36, 41)), we assumed that the movement of mtMCM along ss or dsDNA occurred with enough force to displace the streptavidin-biotin complex (Kd 10–15 M–1 (42)) in its path. As shown in Fig. 3, this was indeed the case. The displacement of streptavidin by mtMCM required ATP hydrolysis, as well as a protein with a functional Walker-A motif.
The displacement of streptavidin from biotinylated DNAs by DNA helicases, such as the phage-T4 gp41 and Dda, has been reported previously (39). The bacterial DnaB was shown to displace the EBNA1 protein-DNA complex (33) but not a streptavidin-biotin DNA complex (32, 33). The mechanism by which certain helicases dislodge streptavidin remains unclear but may depend in part on the distortion introduced into streptavidin upon its impact with a moving helicase. Higher temperatures, as in this case of mtMCM, could also play a role. It should be noted that the structure of the helicases that displace streptavidin vary, i.e. Dda appears to be a monomer (43), phage-T4 gp41 is a hexamer (35), and the mtMCM is a dodecamer (4, 36). Nevertheless, the ability of helicases to remove proteins and other tightly bound molecules was suggested to play a role during DNA transactions including replication, recombination, and repair.
A speculative model of the action of the two helicases studied here is shown in Fig. 5. We suggest that the hexameric spMcm4,6,7 complex binds to the 3'-ssDNA region, which is passed through its central cavity. Translocation in the 3' 5' direction leads to its encounter with the downstream duplex region. If this duplex region contains a 5'-ssDNA overhang of 20 nt or longer (37), the 5'-strand of the duplex is displaced and positioned outside the channel so that 3' 5' movement unwinds the dsDNA. If, as shown in Fig. 5A, the 5' end is not tailed, the spMcm complex encircles both strands of the duplex region. Because both strands are now encircled, further translocation results in no apparent unwinding. However, upon encountering a nick followed by a forked duplex immediately downstream, the right duplex is displaced. The displacement of the right duplex supports the conclusion that translocation along dsDNA has occurred. It is possible that a second hexamer, which binds to the 5'-ssDNA overhang of the right duplex, interacts with the hexamer loaded on the 3'-ssDNA overhang and contributes to the unwinding reaction. The formation of double hexamers on forked structures was proposed for the eukaryal MCM (37) and the bacterial DnaB (44) proteins.
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To date, only two archaeal MCM complexes have been characterized. Although the mtMCM is dodecameric in solution (4, 23–25, 36), the enzyme from S. solfataricus is hexameric (26). Thus, it will be of interest to determine whether an archaeal MCM that is hexameric is capable of translocating along dsDNA and whether the requirements for such movement are similar to those for mtMCM or the eukaryotic enzyme.
A number of suggestions have been proposed for the biological role of dsDNA translocation carried out by the replicative helicases. A model of the action of the eukaryotic MCM complex has been proposed recently (31). In this model, MCM molecules are loaded at the origins by encircling dsDNA through formation of the pre-replication complex. However, rather than remaining at the origin, it was proposed that MCM complexes translocate away from the origin and then act as a rotary pump to drive duplex unwinding at replication factories. This model was proposed to explain the findings in Eukarya that the location of the MCM complex fails to coincide spatially with sites of DNA replication and that the amount of MCM complex present on DNA is much greater (10–100-fold) than the level of the origin recognition complex. The findings reported here suggest that some of the discrepancies noted above may be due, in part, to translocation of the MCM complex along dsDNA, if the loading of the MCM complex results in the passage of dsDNA through its central channel. However, to date we have failed to detect helicase activity with the Mcm2–7 complex or its ability to translocate along dsDNA.
It was also suggested that the movement of the replicative helicase along dsDNA may play a role in preventing DNA unwinding when the helicase encounters damaged regions in DNA that lead to formation of gaps (lagging strand in Eukarya and Archaea and leading strand in bacteria). A switch from an unwinding reaction to a dsDNA transit mode would prevent continued polymerase action. The data presented in Fig. 4 support the possible role for dsDNA movement by both helicases when they encounter a nick present on the leading strand (the translocating strand), as shown in Fig. 4I. When encountering a nick on the lagging strand, the spMcm4/6/7 helicase appears to switch to dsDNA movement whereas the mtMCM complex does not (Fig. 4J). The biological consequences of this difference remain to be explored.
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FOOTNOTES |
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¶ American Cancer Society Research Professor.
|| To whom correspondence should be addressed: University of Maryland Biotechnology Inst., Center for Advanced Research in Biotechnology, 9600 Gudelsky Dr., Rockville, MD 20850. Tel.: 301-738-6294; Fax: 301-738-6255; E-mail: kelman{at}umbi.umd.edu.
1 The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; MCM, minichromosome maintenance; mt, M. thermautotrophicus; sp, Schizosaccharomyces pombe; nt, nucleotide; AMP-PNP, adenosine 5'-(
,-imino)triphosphate; ATPS, adenosine 5'-O-(thiotriphosphate).
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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 A. Devault, E. Gueydon, and E. Schwob Interplay between S-Cyclin-dependent Kinase and Dbf4-dependent Kinase in Controlling DNA Replication through Phosphorylation of Yeast Mcm4 N-Terminal Domain Mol. Biol. Cell, May 1, 2008; 19(5): 2267 - 2277. [Abstract] [Full Text] [PDF]
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 N. Sakakibara, R. Kasiviswanathan, E. Melamud, M. Han, F. P. Schwarz, and Z. Kelman Coupling of DNA binding and helicase activity is mediated by a conserved loop in the MCM protein Nucleic Acids Res., March 27, 2008; 36(4): 1309 - 1320. [Abstract] [Full Text] [PDF]
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E. Rothenberg, M. A. Trakselis, S. D. Bell, and T. Ha MCM Forked Substrate Specificity Involves Dynamic Interaction with the 5'-Tail J. Biol. Chem., November 23, 2007; 282(47): 34229 - 34234. [Abstract] [Full Text] [PDF]
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M. L. Bochman and A. Schwacha Differences in the Single-stranded DNA Binding Activities of MCM2-7 and MCM467: MCM2 AND MCM5 DEFINE A SLOW ATP-DEPENDENT STEP J. Biol. Chem., November 16, 2007; 282(46): 33795 - 33804. [Abstract] [Full Text] [PDF]
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J.-H. Shin, T. J. Santangelo, Y. Xie, J. N |
