The Hexameric Helicase DnaB Adopts a Nonplanar Conformation during Translocation

Itsathitphaisarn, Ornchuma; Wing, Richard A.; Eliason, William K.; Wang, Jimin; Steitz, Thomas A.

doi:10.1016/j.cell.2012.09.014

Cited by 209 publications

(377 citation statements)

References 51 publications

Supporting

Mentioning

334

Contrasting

Unclassified

Order By: Relevance

“…ATP signifies the Walker A site and R signifies the arginine finger. Crystal structure analysis of BPV E1 (12), E. coli DnaB (13), and E. coliring (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

“…This report uses cryo-EM single-particle reconstruction to image CMG that motored to a block site at a forked junction, enabling direct visualization of DNA threading through CMG. (12), Rho (3), and DnaB (13), representatives of SF3, SF5, and SF4 helicases, respectively. The polarity of DNA relative to the ATP sites is informative.…”

Section: Significancementioning

confidence: 99%

“…1), yet they translocate on DNA in opposite directions (3). Hence, in BPV E1 helicase the N-tier is pushed from behind by the C-tier motor, whereas in Rho and DnaB the C-tier motor is in front and pulls the N-tier ring behind during translocation (3,12,13). The object of this report is to determine whether the DNA orientation through the N-tier and C-tier rings of CMG is different or similar to the hexameric helicases of other superfamilies.…”

Section: Significancementioning

confidence: 99%

See 2 more Smart Citations

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.

185

299

View full text Add to dashboard Cite

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...

show abstract

“…ATP signifies the Walker A site and R signifies the arginine finger. Crystal structure analysis of BPV E1 (12), E. coli DnaB (13), and E. coliring (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

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.

185

299

View full text Add to dashboard Cite

show abstract

“…A similar mechanism, albeit with an opposite polarity, was described for an ssRNA translocase: the Escherichia coli transcription termination factor Rho (12) (also reviewed in ref. 13) and for the bacterial hexameric helicase DnaB, although in this case the authors propose a two-nucleotide translocation step (14). In addition, the sequential hydrolysis model requires subunits to switch conformations at the nucleotide-binding site during the hydrolysis cycle, as described in the ClpX AAA+ unfoldase (15).…”

mentioning

confidence: 99%

Dynamic look at DNA unwinding by a replicative helicase

Lee

Syed

Enemark

et al. 2014

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

View full text Add to dashboard Cite

A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.ATPase | molecular motors D NA replication is the most fundamental of all of life's processes. One of the key requisites in the initiation of replication is the separation of the two strands of the double helix, which is carried out by a hexameric helicase. Despite their prominent roles in biology, some of the basic aspects of these helicases, whether they use a strand-exclusion mechanism or whether they translocate along double-stranded DNA, for example, have been subjects of considerable debate (1, 2). Viral replicative helicases, such as SV40 Large-T antigen (LTag) and papillomavirus E1, have provided the opportunity to study some of these basic features largely owing to their homohexameric architecture. These viral helicases recognize their respective origin of DNA replication (ori) through their dsDNA-binding domains (DBDs) and assemble in a stepwise fashion, ultimately forming double-hexameric (DH) structures on their ori and unwind the DNA bidirectionally.E1 consists of an N-terminal domain, a DBD, an oligomerization domain (OD), a helicase/ATPase domain (HD), and a C-terminal acidic tail (Fig. 1A). Biochemical and structural data have demonstrated that the DBDs bound to the pseudopalindromic E1 binding site are at the center of the double hexamer and that the helicase domains that bind to the flanks of the ori are on either end in a head-to-head arrangement (3, 4). This arrangement is supported by EM studies of LTag that show a dumbbell-shaped structure for the DH (5, 6), with each half of the dumbbell containing two lobes: a larger HD outer lobe and a smaller DBD inner lobe. The assembled DH appears to place the HD of E1 proximal to the dsDNA to be unwound.However, in the structure of the E1 helicase with ssDNA and Mg 2+ -ADP, the ssDNA is oriented such that the N-terminal part of the polypeptide (the OD) is closest to the 5′ end of the DNA (7). The 3′→5′ polarity of E1 helicase indicates that the translocating helicase moves with the N-terminal OD leading and the C-terminal helicase domain trailing, or alternatively, that the DNA is pumped through the helicase from the OD side...

show abstract

“…To capture a view of the proteasome mid-catalysis, Ding et al determined the structure of the proteasome in the presence of the ATP analog ADP-AlFx. In other ATPases [4,5], provision of ADP-AlFx locks the ATPase motor in a transitionlike state of the ATP hydrolysis cycle, and thus, their structure likely reveals the proteasome's ATPase motor midstroke. In their model, the ATPase ring of the proteasome is in a very different conformation than that observed in all previous dwell-phase structures, and provides new insights into the nature of the ATPase cycle and its relation to substrate unfolding.…”

mentioning

confidence: 99%

Proteasomes, caught in the act

Tomko

2017

Cell Res

View full text Add to dashboard Cite

Although energy-dependent protein destruction by the proteasome has been known for over 30 years, how this intricate molecular machine uses ATP to power protein degradation has remained very poorly understood. In a recently published paper, Ding et al. present a snapshot of the proteasome mid-catalysis, yielding new and unexpected insights into the catalytic mechanism of this ATPpowered multisubunit machine.Cells rely on large multisubunit molecular machines to conduct many complex biological processes, such as protein synthesis, folding, and degradation. In eukaryotes, most regulated protein degradation is conducted by the proteasome, a massive 66-subunit ATP-dependent protease complex. Although recent advances in cryo-electron microscopy (cryo-EM) have yielded an onslaught of information about the proteasome, none have yet revealed how ATP drives the proteasome's catalytic cycle. By pharmacologically trapping the proteasome in a transition state, Ding et al. [1] provide a snapshot of the proteasome's molecular motor in action. This snapshot yields answers as well as poses new questions regarding the catalytic mechanism of this fascinating multisubunit machine.The proteasome consists of three subcomplexes ( Figure 1A), the barrelshaped core particle (CP), which houses the peptidase active sites; the ringshaped regulatory particle (RP) base, which abuts the ends of the CP; and the RP lid, which embraces the base and CP and hovers over their central pores. These subcomplexes function together much like an assembly line to bind the

show abstract

The Hexameric Helicase DnaB Adopts a Nonplanar Conformation during Translocation

Cited by 209 publications

References 51 publications

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

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

Dynamic look at DNA unwinding by a replicative helicase

Proteasomes, caught in the act

Contact Info

Product

Resources

About