DNA helicase from calf thymus. Purification to apparent homogeneity and biochemical characterization of the enzyme

We have purified a DNA helicase from calf thymus to apparent homogeneity by monitoring the activity with a strand displacement assay. DNA helicase followed the DNA polymerase alpha-primase complex through chromatography on phosphocellulose and hydroxylapatite. Separation from DNA polymerase alpha-primase complex as well as from the bulk of another DNA-dependent ATPase was achieved on heparin-Sepharose. Further purification steps included ATP-agarose and fast protein liquid chromatography-Mono S. A 47-kDa polypeptide cosedimented with the DNA helicase activity in a glycerol gradient as well as in gel filtration on Superose 6. The calf thymus DNA helicase had a sedimentation coefficient of 4-7 S and Stokes radius of about 45 A suggesting that the enzyme might be monomer in its functional form. DNA helicase activity requires a divalent cation with Mg2+ being more efficient than Mn2+ or Ca2+. Hydrolysis of ATP is required since the two nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenylyl (beta, gamma-methylene)diphosphonate cannot substitute for ATP or dATP in the displacement reaction. Calf thymus DNA helicase is able to use ATP, dATP, dideoxy-ATP, CTP, and dCTP with Km for ATP and dATP of 0.2 and 0.25 mM, respectively. The enzyme can displace a fragment of 24 bases completely in an enzyme concentration- and time-dependent manner. The DNA helicase appears to bind to single-stranded DNA and to move to single-strand double-strand transition. The directionality of unwinding is 3'----5' with respect to the single-stranded DNA to which the enzyme is bound.     

Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase

We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.     

Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA

Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability. The WS protein (WRN) possesses 3′→5′ DNA helicase and associated ATPase activities, as well as 3′→5′ DNA exonuclease activity. Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity. It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype. WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme. We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD. We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis. Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation. We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.     

Potent inhibition of DNA unwinding and ATPase activities of pea DNA helicase 45 by DNA-binding agents

Pea DNA helicase 45 (PDH45) is an ATP-dependent DNA unwinding enzyme, with intrinsic DNA-dependent ATPase activity [Plant J. 24 (2000) 219]. We have determined the effect of various DNA-binding agents, such as daunorubicin, ethidium bromide, ellipticine, cisplatin, nogalamycin, actinomycin C1, and camptothecin on the DNA unwinding and ATPase activities of the plant nuclear DNA helicase PDH45. The results show that all the agents except actinomycin C1, and camptothecin inhibited the helicase (apparent K(i) values ranging from 1.5 to 7.0 microM) and ATPase (apparent K(i) values ranging from 2.5 to 11.9 microM) activities. This is the first study to show the effect of various DNA-binding agents on the plant nuclear helicase and also first to demonstrate inhibition of any helicase by cisplatin. Another striking finding that the actinomycin C1 and ellipticine act differentially on PDH45 as compared to pea chloroplast helicase suggests that the mechanism of DNA unwinding could be different in nucleus and chloroplast. These results suggest that the intercalation of the inhibitors into duplex DNA generates a complex that impedes translocation of PDH45, resulting in both the inhibitions of unwinding activity and ATP hydrolysis. This study would be useful to obtain a better understanding of the mechanism of plant nuclear DNA helicase unwinding and the mechanism by which these agents can disturb genome integrity.     

Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex

DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB x DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [Kd = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATPgammaS, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATPgammaS, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1:1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB6 x DnaC6 dodecameric complex. Our results demonstrate that the DnaB6 x DnaC6 complex bound ssDNA with a high affinity [Kd = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATPgammaS or ADP, binding of ssDNA by the DnaB6 x DnaC6 complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB6 x DnaC6 complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.     

A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA.

A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.     

DNA synthesis at a fork in the presence of DNA helicases

In a mixture of Escherichia coli DNA polymerase III holoenzyme, single-strand-binding protein, artificially forked lambda bacteriophage DNA with primer annealed to the leading side of the fork, dNTPs and ATP, DNA synthesis is enhanced by helicase II, less so by helicases, I, III or rep protein of E. coli or T4 phage helicase. The effect of helicase II depends on ATP, it is enhanced by helicase III, and it is not observed using DNA polymerase I or T4 DNA polymerase. In the absence of dNTPs helicase II is less active than helicase I or T4 helicase in unwinding the forked DNA. We believe that helicase II both shifts the forks and stimulates DNA polymerase III. The results support the conclusion derived from previous studies that helicase II is part of the DNA-synthesizing system of E. coli.     

Biochemical analysis of the intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity

Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood. Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme. In this study, we have expressed and isolated the mouse Mcm2, 4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated. The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex. Biochemical analyses of these mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex. The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated.     

DNA unwinding is an Mcm complex-dependent and ATP hydrolysis-dependent process

Minichromosome maintenance proteins (Mcm) are essential in all eukaryotes and are absolutely required for initiation of DNA replication. The eukaryotic and archaeal Mcm proteins have conserved helicase motifs and exhibit DNA helicase and ATP hydrolysis activities in vitro. Although the Mcm proteins have been proposed to be the replicative helicase, the enzyme that melts the DNA helix at the replication fork, their function during cellular DNA replication elongation is still unclear. Using nucleoplasmic extract (NPE) from Xenopus laevis eggs and six purified polyclonal antibodies generated against each of the Xenopus Mcm proteins, we have demonstrated that Mcm proteins are required during DNA replication and DNA unwinding after initiation of replication. Quantitative depletion of Mcms from the NPE results in normal replication and unwinding, confirming that Mcms are required before pre-replicative complex assembly and dispensable thereafter. Replication and unwinding are inhibited when pooled neutralizing antibodies against the six different Mcm2-7 proteins are added during NPE incubation. Furthermore, replication is blocked by the addition of the Mcm antibodies after an initial period of replication in the NPE, visualized by a pulse of radiolabeled nucleotide at the same time as antibody addition. Addition of the cyclin-dependent kinase 2 inhibitor p21(cip1) specifically blocks origin firing but does not prevent helicase action. When p21(cip1) is added, followed by the non-hydrolyzable analog ATPgammaS to block helicase function, unwinding is inhibited, demonstrating that plasmid unwinding is specifically attributable to an ATP hydrolysis-dependent function. These data support the hypothesis that the Mcm protein complex functions as the replicative helicase.     

Rep protein as a helicase in an active, isolatable replication fork of duplex phi X174 DNA

Rep protein as a helicase combines its actions with those of gene A protein and single-stranded DNA binding protein to separate the strands of phi X174 duplex DNA and thereby can generate and advance a replication fork (Scott, J. F., Eisenberg, S., Bertsch, L. L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 193-197). Tritium-labeled rep protein is bound in an active gene A protein. phi X174 closed circular duplex supercoiled DNA complex in a 1:1 ratio. Catalytic separation of the strands of the duplex by rep protein, as measured by incorporation of tritium-labeled single-stranded DNA binding protein, requires ATP at a Km value of 8 microM, and hydrolyzes two molecules of ATP for every base pair melted. When coupled to replication in the synthesis of single-strand viral circles, a "looped" rolling-circle intermediate is formed that can be isolated in an active form containing gene A protein, rep protein, single-stranded DNA binding protein, and DNA polymerase III holoenzyme. Unlike the binding of rep protein to single-stranded DNA, where its ATPase activity is distributive, binding to the replicating fork is not affected by ATP, further suggesting a processive action linked to gene A protein. Limited tryptic hydrolysis of rep protein abolishes its replicative activity without affecting significantly its binding of ATP and its ATPase action on single-stranded DNA. These results augment earlier findings by describing the larger role of rep proteins as a helicase, linked in a complex ith other proteins, at the replication fork of a duplex DNA.     

The study of interactions between DNA and PcrA DNA helicase by using targeted molecular dynamic simulations

DNA helicases are important enzymes involved in all aspects of nucleic acid metabolism, ranging from DNA replication and repair to recombination, rescue of stalled replication and translation. DNA helicases are molecular motors. Through conformational changes caused by ATP hydrolysis and binding, they move along the template double helix, break the hydrogen bonds between the two strands and separate the template chains, so that the genetic information can be accessed. In this paper, targeted molecular dynamic simulations were performed to study the important interactions between DNA and PcrA DNA helicase, which can not be observed from the crystal structures. The key residues on PcrA DNA helicase that have strong interactions with both double stranded DNA (ds-DNA) and single stranded DNA (ss-DNA) have been identified, and it was found that such interactions mostly exist between the protein and DNA backbone, which indicates that the translocation of PcrA is independent of the DNA sequence. The simulations indicate that the ds-DNA is separated upon ATP rebinding, rather than ATP hydrolysis, which suggests that the two strokes in the mechanism have two different major roles. Firstly, in the power stroke (ATP hydrolysis), most of the translocations of the bases from one pocket to the next occur. In the relaxation stroke (ATP binding), most of the ‘work’ is being done to ‘melt’ the DNA at the separation fork. Therefore, we propose a mechanism whereby the translocation of the ss-DNA is powered by ATP hydrolysis and the separation of the ds-DNA is powered by ATP binding.     

Electron microscopic observation and single-stranded DNA binding activity of the Mcm4,6,7 complex

Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.     

The T4 phage SF1B helicase Dda is structurally optimized to perform DNA strand separation

Helicases move on DNA via an ATP binding and hydrolysis mechanism coordinated by well-characterized helicase motifs. However, the translocation along single-stranded DNA (ssDNA) and the strand separation of double-stranded (dsDNA) may be loosely or tightly coupled. Dda is a phage T4 SF1B helicase with sequence homology to the Pif1 family of helicases that tightly couples translocation to strand separation. The crystal structure of the Dda-ssDNA binary complex reveals a domain referred to as the "pin" that was previously thought to remain static during strand separation. The pin contains a conserved phenylalanine that mediates a transient base-stacking interaction that is absolutely required for separation of dsDNA. The pin is secured at its tip by protein-protein interactions through an extended SH3 domain thereby creating a rigid strut. The conserved interface between the pin and the SH3 domain provides the mechanism for tight coupling of translocation to strand separation.     

The SV40 large T-antigen helicase can unwind four stranded DNA structures linked by G-quartets.

We describe a novel activity of the SV40 large T-ag helicase, the unwinding of four stranded DNA structures linked by stacked G-quartets, namely stacked groups of four guanine bases bound by Hoogsteen hydrogen bonds. The structures unwound by the helicase were of two types: (i) quadruplexes comprising four parallel strands that were generated by annealing oligonucleotides including clustered G residues in a buffer containing Na+ions. Each parallel quadruplex consisted of four oligonucleotide molecules. (ii) Complexes comprising two parallel and two antiparallel strands that were generated by annealing the above oligonucleotides in a buffer containing K+ions. Each antiparallel complex consisted of two folded oligonucleotide molecules. Unwinding of these unusual DNA structures by the T-ag was monitored by gel electrophoresis. The unwinding process required ATP and at least one single stranded 3'-tail extending beyond the four stranded region. These data indicated that the T-ag first binds the 3'-tail and moves in a 3'-->5'direction, using energy provided by ATP hydrolysis; then it unwinds the four stranded DNA into single strands. This helicase activity may affect processes such as recombination and telomere extension, in which four stranded DNA could play a role.     

Escherichia coli DNA helicases: mechanisms of DNA unwinding

DNA helicases are ubiquitous enzymes that catalyse the unwinding of duplex DNA during replication, recombination and repair. These enzymes have been studied extensively; however, the specific details of how any helicase unwinds duplex DNA are unknown. Although it is clear that not all helicases unwind duplex DNA in an identical way, many helicases possess similar properties, which are thus likely to be of general importance to their mechanism of action. For example, since helicases appear generally to be oligomeric enzymes, the hypothesis is presented in this review that the functionally active forms of DNA helicases are oligomeric. The oligomeric nature of helicases provides them with multiple DNA-binding sites, allowing the transient formation of ternary structures, such that at an unwinding fork, the helicase can bind either single-stranded and duplex DNA simultaneously or two strands of single-stranded DNA. Modulation of the relative affinities of these binding sites for single-stranded versus duplex DNA through ATP binding and hydrolysis would then provide the basis for a cycling mechanism for processive unwinding of DNA by helicases. The properties of the Escherichia coli DNA helicases are reviewed and possible mechanisms by which helicases might unwind duplex DNA are discussed in view of their oligomeric structures, with emphasis on the E. coli Rep, RecBCD and phage T7 gene 4 helicases.     

A DNA helicase from Xenopus laevis ovaries

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.     

Role of ATP hydrolysis in the DNA translocase activity of the bovine papillomavirus (BPV-1) E1 helicase

The E1 protein of bovine papillomavirus type-1 is the viral replication initiator protein and replicative helicase. Here we show that the C-terminal ~300 amino acids of E1, that share homology with members of helicase superfamily 3 (SF3), can act as an autonomous helicase. E1 is monomeric in the absence of ATP but assembles into hexamers in the presence of ATP, single-stranded DNA (ssDNA) or both. A 16 base sequence is the minimum for efficient hexamerization, although the complex protects ~30 bases from nuclease digestion, supporting the notion that the DNA is bound within the protein complex. In the absence of ATP, or in the presence of ADP or the non–hydrolysable ATP analogue AMP–PNP, the interaction with short ssDNA oligonucleotides is exceptionally tight (T_1/2 > 6 h). However, in the presence of ATP, the interaction with DNA is destabilized (T_1/2 ~60 s). These results suggest that during the ATP hydrolysis cycle an internal DNA-binding site oscillates from a high to a low-affinity state, while protein–protein interactions switch from low to high affinity. This reciprocal change in protein–protein and protein–DNA affinities could be part of a mechanism for tethering the protein to its substrate while unidirectional movement along DNA proceeds.     

Simian virus 40 large T antigen untwists DNA at the origin of DNA replication.

Simian virus 40 large tumor antigen (SV40 T antigen) untwists DNA at the SV40 replication origin. In the presence of ATP, T antigen shifted the average linking number of an SV40 origin-containing plasmid topoisomer distribution. The loss of up to two helical turns was detected. The reaction required the presence of the 64-base pair core origin of replication containing T antigen DNA binding site II; binding site I had no effect on the untwisting reaction. The presence of human single-stranded DNA binding protein (SSB) slightly reduced the degree of untwisting in the presence of ATP. ATP hydrolysis was not required since untwisting occurred in the presence of nonhydrolyzable analogs of ATP. However, in the presence of a nonhydrolyzable analog of ATP, the requirement for the SV40 origin sequence was lost. The origin requirement for DNA untwisting was also lost in the absence of dithiothreitol. The origin-specific untwisting activity of T antigen is distinct from its DNA helicase activity, since helicase activity does not require the SV40 origin but does require ATP hydrolysis. The lack of a requirement for SSB or ATP hydrolysis and the reduction in the pitch of the DNA helix by just a few turns at the replication origin distinguishes this reaction from the T antigen-mediated DNA unwinding reaction, which results in the formation of a highly underwound DNA molecule. Untwisting occurred without a lag after the start of the reaction, whereas unwound DNA was first detected after a lag of 10 min. It is proposed that the formation of a multimeric T antigen complex containing untwisted DNA at the SV40 origin is a prerequisite for the initiation of DNA unwinding and replication.     

Mutations altering the interplay between GkDnaC helicase and DNA reveal an insight into helicase unwinding

Replicative helicases are essential molecular machines that utilize energy derived from NTP hydrolysis to move along nucleic acids and to unwind double-stranded DNA (dsDNA). Our earlier crystal structure of the hexameric helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in complex with single-stranded DNA (ssDNA) suggested several key residues responsible for DNA binding that likely play a role in DNA translocation during the unwinding process. Here, we demonstrated that the unwinding activities of mutants with substitutions at these key residues in GkDnaC are 2-4-fold higher than that of wild-type protein. We also observed the faster unwinding velocities in these mutants using single-molecule experiments. A partial loss in the interaction of helicase with ssDNA leads to an enhancement in helicase efficiency, while their ATPase activities remain unchanged. In strong contrast, adding accessory proteins (DnaG or DnaI) to GkDnaC helicase alters the ATPase, unwinding efficiency and the unwinding velocity of the helicase. It suggests that the unwinding velocity of helicase could be modulated by two different pathways, the efficiency of ATP hydrolysis or protein-DNA interaction.     

Molecular architecture of a multifunctional MCM complex

DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase-primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPγS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.