Twin DNA Pumps of a Hexameric Helicase Provide Power to Simultaneously Melt Two Duplexes

Kaplan, Daniel L.; O'Donnell, Mike

doi:10.1016/j.molcel.2004.06.039

Cited by 57 publications

(71 citation statements)

References 36 publications

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“…Given this poor binding, the ⌬wedge protein displays surprisingly robust unwinding activity with J12 and RF, although concentrations far in excess of those required for the wild-type protein are required to see substantial unwinding of the substrate. It is likely that the unwinding is simply caused by trying to pull a branched molecule through a DNA translocation channel that is only large enough to take a single DNA duplex, in a similar manner to that reported for the hexameric DnaB helicase (4,5). This suggests that the translocation activity is relatively unaffected by the loss of the wedge domain in a manner similar to substitutions at Phe 96 and Phe 97 .…”

Section: Discussionmentioning

confidence: 70%

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DNA Binding by the Substrate Specificity (Wedge) Domain of RecG Helicase Suggests a Role in Processivity

Briggs¹,

Mahdi²,

Qin

et al. 2005

Journal of Biological Chemistry

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RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.Helicases are ubiquitous enzymes essential in all stages of DNA and RNA metabolism, including replication, transcription, recombination, and repair (1, 2). They are motor proteins that generally translocate along one or both strands of dsDNA, 1 dsRNA, or a DNA-RNA hybrid in an ATP-dependent manner and separate some or all parts of the molecule into its component strands. Some enzymes are highly processive, driving strand separation for long distances without dissociating from the template, a property that is especially important for enzymes involved in chromosome replication (3).A variety of mechanisms has evolved to ensure processivity. The prokaryotic helicase DnaB, for example, forms a hexameric ring that completely encircles the DNA, which allows onedimensional motion while preventing dissociation (4, 5). Such a strategy is common to many of the hexameric helicases (6). Other helicases are thought to achieve the same effect by interacting with the "sliding clamp" that encircles the DNA strands during replication. Examples include Wrn and Rrm3, which have been shown to have enhanced processivity in the presence of PCNA (7,8). The RecC component of the RecBCD helicase has no helicase activity in itself, but its role may be similar to a sliding clamp, preventing dissociation of the RecBCD complex from the DNA (9 -11).A second strategy is for the helicase to have two binding sites, such that one site can release and move along the strand while the second stays bound. This can be achieved by having a dimeric structure, with one binding site per monomer and has led to the proposal of the "hand over hand" or "rolling" method. Rep helicase is thought to translocate using this mechanism (12). The Rep dimer binds to DNA, with one monomer behind the other. ATP hydrolysis alters the conformation of the helicase, radically changing the position of one monomer relative to the other, as well as causing a change in its nucleic acid binding properties (12)(13)(14). This movement means the second monomer can now bind the DNA in front of the first monomer, and thus translocation i...

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Section: Discussionmentioning

confidence: 70%

“…The prokaryotic helicase DnaB, for example, forms a hexameric ring that completely encircles the DNA, which allows onedimensional motion while preventing dissociation (4,5). Such a strategy is common to many of the hexameric helicases (6).…”

mentioning

confidence: 99%

DNA Binding by the Substrate Specificity (Wedge) Domain of RecG Helicase Suggests a Role in Processivity

Briggs¹,

Mahdi²,

Qin

et al. 2005

Journal of Biological Chemistry

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“…Furthermore, in view of the conclusion that only a single strand of DNA transits through the central channel of T-ag, it is of interest that the bovine papillomavirus (BPV) E1 helicase assembles around a single strand of DNA whose 3Ј end is directed towards the C terminus (25). Moreover, the steric exclusion model for DnaB and MCM (38,39) as well as Rho (87) suggests that one DNA strand transits through the central channels, while the second strand passes outside the rings. Similarly, models suggest that the T7 helicase ring surrounds the 5Ј strand in its central channel, thus accounting for its 5Ј-to-3Ј polarity, while the 3Ј strand is routed over the external surface (2,24,32).…”

Section: Discussionmentioning

confidence: 99%

Model for T-Antigen-Dependent Melting of the Simian Virus 40 Core Origin Based on Studies of the Interaction of the Beta-Hairpin with DNA

Kumar

Meinke

Reese

et al. 2007

J Virol

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The interaction of simian virus 40 (SV40) T antigen (T-ag) with the viral origin has served as a model for studies of site-specific recognition of a eukaryotic replication origin and the mechanism of DNA unwinding. These studies have revealed that a motif termed the "beta-hairpin" is necessary for assembly of T-ag on the SV40 origin. Herein it is demonstrated that residues at the tip of the "beta-hairpin" are needed to melt the origin-flanking regions and that the T-ag helicase domain selectively assembles around one of the newly generated single strands in a manner that accounts for its 3-to-5 helicase activity. Furthermore, T-ags mutated at the tip of the "beta-hairpin" are defective for oligomerization on duplex DNA; however, they can assemble on hybrid duplex DNA or single-stranded DNA (ssDNA) substrates provided the strand containing the 3 extension is present. Collectively, these experiments indicate that residues at the tip of the beta-hairpin generate ssDNA in the core origin and that the ssDNA is essential for subsequent oligomerization events.Replication of DNA is a fundamental biological process that has been highly conserved (36). However, at the molecular level, many steps during DNA replication are not well understood. For example, our understanding of initiation of replication in higher eukaryotes is limited owing to the failure to identify sequences that serve as origins of replication (28). An additional complexity is that while a number of factors needed to initiate eukaryotic DNA replication have been identified, the functions of many of these proteins have yet to be established (36, 86).Therefore, fundamental issues related to the initiation of DNA replication in eukaryotes are being addressed using the relatively simple DNA tumor viruses (78). The DNA sequences that serve as viral origins of replication have been characterized extensively (reviewed in references 10 and 26). For instance, the simian virus 40 (SV40) core origin is 64 bp long and contains three regions, including a central region containing four GAGGC pentanucleotides arranged as inverted repeats and two flanking regions termed the early palindrome (EP) and the AT-rich region. Viral origins are recognized by virally encoded proteins termed initiators; the SV40-encoded initiator is a multidomain, multifunctional protein termed the large T antigen (T-ag) (reviewed in references 14, 26, and 73). T-ag belongs to helicase superfamily III and is also a member of the AAAϩ (ATPase associated with cellular activities) family of proteins (31). It contains an N-terminal J domain, a central origin binding domain (T-ag-obd), and a C-terminal helicase domain (reviewed in references 14, 26, and 73). The determination of much of the structure of T-ag, including the J domain (40), T-ag-obd (49, 54), and the helicase domain (27, 42), has greatly expanded our understanding of T-ag and its assembly on the origin. For example, the T-ag-obd has been shown to form an open ring structure in the crystal, with a positively charged central channel that i...

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“…Thus, the head-to-head geometry, and N-tier to C-tier orientation during translocation, combine to provide a quality control mechanism that requires both CMGs to encircle ssDNA before either leaves the origin. Head-to-head hexameric helicases that encircle dsDNA have also been demonstrated to facilitate DNA melting (42), and thus the head-on orientation of two CMGs may provide energy for the initial unwinding of dsDNA at origins.…”

Section: Implications Of N-tier Ahead Of C-tier During Cmg Translocatmentioning

confidence: 99%

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

Georgescu

Yuan

Bai

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

182

299

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The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5′-3′ through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol e below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle doublestranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.CMG helicase | DNA replication | DNA polymerase | origin initiation | replisome R eplicative helicases are hexameric rings in all domains of life (1-3). In bacteria and archaea, the replicative helicase is a homohexamer and encircles single-strand (ss) DNA at a replication fork. Some viral and phage replicative helicases are also ring-shaped hexamers, including bovine papilloma virus (BPV) E1, simian virus 40 (SV40) large T-antigen (T-Ag), and the T4 and T7 phage helicases. Unlike other replicative helicases, the eukaryotic replicative Mcm2-7 helicase is composed of six nonidentical but homologous Mcm subunits that become activated upon assembly with five accessory factors (Cdc45 and GINS tetramer) to form the 11-subunit CMG (Cdc45, Mcm2-7, GINS) (4-6). Numerous studies have outlined the process that forms CMG at origins in which the Mcm2-7 heterohexamer is loaded onto DNA as an inactive double hexamer in G1 phase, and becomes activated in S phase by several initiation proteins and cell-cycle kinases that assemble Cdc45 and GINS onto Mcm2-7 to form the active CMG helicases (7-9).Helicases assort into six superfamilies (SF1-SF6) based on sequence alignments (10). The SF1 and SF2 helicases are generally monomeric and the SF3-SF6 helicases are hexameric rings used in DNA replication and other processes. The bacterial SF4 and SF5 helicases contain RecA-based motors and translocate 5′-3′, whereas the eukarytic SF3 and SF6 helicases contain AAA+ (ATPases associated with diverse cellular activities)-based motors and translocate 3′-5′ (3, 10). Examples of well-studied hexameric helicases include t...

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Twin DNA Pumps of a Hexameric Helicase Provide Power to Simultaneously Melt Two Duplexes

Cited by 57 publications

References 36 publications

DNA Binding by the Substrate Specificity (Wedge) Domain of RecG Helicase Suggests a Role in Processivity

DNA Binding by the Substrate Specificity (Wedge) Domain of RecG Helicase Suggests a Role in Processivity

Model for T-Antigen-Dependent Melting of the Simian Virus 40 Core Origin Based on Studies of the Interaction of the Beta-Hairpin with DNA

Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation

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