ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

Schuck, Stephen; Stenlund, Arne

doi:10.1128/jvi.02432-06

Cited by 15 publications

(17 citation statements)

References 40 publications

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“…We have shown that E1 binds to a dsDNA ori probe, forming a double trimer (DT); this DT is converted into a DH, which unwinds the template in the presence of E. coli single-strand DNA-binding protein (SSB), resulting in ssDNA+SSB complexes that can be detected by EMSA. This reaction is dependent on the formation of the E1 DH, which in turn is dependent on the centrally located E1 binding sites (BSs) and sequences on the flanks of the E1 BS (3,21,22). If both strands of the DNA pass through the central channel of the helicase, we expect that a streptavidin attached to either the 5′ or 3′ end of the DNA would be displaced, whereas if only one strand passes through the central channel, only streptavidin attached to that strand would be displaced.…”

Section: Resultsmentioning

confidence: 99%

Dynamic look at DNA unwinding by a replicative helicase

Lee

Syed

Enemark

et al. 2014

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

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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...

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Section: Resultsmentioning

confidence: 99%

Dynamic look at DNA unwinding by a replicative helicase

Lee

Syed

Enemark

et al. 2014

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

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“…For DnaA and systems like the eukaryotic Origin Recognition Complex (ORC), both of which belong to the same AAAϩ subgroup, the conformation, assembly, and origin recognition properties of the initiator all can be altered by ATP (17, 31, 48 -51). For the more distantly related SFIII helicases, a group of AAAϩ proteins that serve as initiators for certain dsDNA viruses, ATP is required for DNA melting as it is for DnaA (52)(53)(54)(55)(56).…”

Section: Discussionmentioning

confidence: 99%

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

Duderstadt

Mott

Crisona

et al. 2010

Journal of Biological Chemistry

134

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The initiation of DNA replication requires the melting of chromosomal origins to provide a template for replisomal polymerases. In bacteria, the DnaA initiator plays a key role in this process, forming a large nucleoprotein complex that opens DNA through a complex and poorly understood mechanism. Using structure-guided mutagenesis, biochemical, and genetic approaches, we establish an unexpected link between the duplex DNA-binding domain of DnaA and the ability of the protein to both self-assemble and engage singlestranded DNA in an ATP-dependent manner. Intersubunit cross-talk between this domain and the DnaA ATPase region regulates this link and is required for both origin unwinding in vitro and initiator function in vivo. These findings indicate that DnaA utilizes at least two different oligomeric conformations for engaging single-and double-stranded DNA, and that these states play distinct roles in controlling the progression of initiation.

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“…The transformation of this species to the active double-hexamer correlates with 'melting' activity and depends upon an aromatic residue on the DNA-binding hairpin of the helicase [42 ]. The melting activity is ATP-dependent [91 ], and could potentially utilize the same molecular motions described for the formed hexamer operating in a limited, local manner. Additional structural snapshots and biochemical investigation will address these questions.…”

Section: Concluding Remarks -Future Challengesmentioning

confidence: 99%

On helicases and other motor proteins

Enemark

Joshua‐Tor

2008

Current Opinion in Structural Biology

192

238

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Helicases are molecular machines that utilize energy derived from ATP hydrolysis to move along nucleic acids and to separate base-paired nucleotides. The movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. Recent structural data for the hexameric E1 helicase of papillomavirus in complex with single-stranded DNA and MgADP has provided a detailed atomic and mechanistic picture of its ATP-driven DNA translocation. The structural and mechanistic features of this helicase are compared with the hexameric helicase prototypes T7gp4 and SV40 T-antigen. The ATP-binding site architectures of these proteins are structurally similar to the sites of other prototypical ATP-driven motors such as F1-ATPase, suggesting related roles for the individual site residues in the ATPase activity. IntroductionHelicases are essential enzymes that unwind duplex DNA, RNA, or DNA-RNA hybrids. This unwinding is driven by consumption of input energy that is harnessed to separate base-paired oligonucleotides and also to maintain a unidirectional advancement of the helicase upon the nucleic acid substrate. This translocation can alternatively be described as an immobile helicase pumping nucleic acid. The energy for these transformations is derived from the hydrolysis of nucleotide triphosphate (NTP). Helicases can be depicted as an internal combustion engine with each individual NTPase site serving as one cylinder. Each individual cylinder follows a defined series of events: injection (ATP binding), compression (optimally positioning the site for hydrolysis), combustion (ATP hydrolysis/work generation), and exhaust (ADP and phosphate release). In a helicase, the individual combustion cylinders coordinate these actions to carry out the repetitive mechanical operation of prying open base pairs and/or actively translocating with respect to the nucleic acid substrate. Many other molecular motors utilize similar engines to carry out multiple diverse functions such as translocation of peptides in the case of ClpX, movement along cellular structures in the case of dyenin, and rotation about an axle as in F 1 -ATPase.Based upon conserved sequence motifs, helicases have been classified into six superfamilies [1,2 ]. An extensive review of these superfamilies has been provided recently [3 ]. Superfamily 1 (SF1) and superfamily 2 (SF2) helicases are very prevalent, generally monomeric, and participate in several diverse DNA and RNA manipulations. The other helicase superfamilies form hexameric rings (reviewed in [4]), as demonstrated by biochemistry [5][6][7][8] and electron microscopy studies [9][10][11][12][13][14][15][16][17], and often participate at the replication fork. All of these helicases bind and hydrolyze NTP at the interface between two recA-like domains. The binding site consists of a Walker A (P-loop) and a Walker B motif from the first domain and other elements such as an arginine finger from the other domain. The SF1 and SF2 helicases contain two recAlike domains coupled by a short linker, and t...

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ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

Cited by 15 publications

References 40 publications

Dynamic look at DNA unwinding by a replicative helicase

Dynamic look at DNA unwinding by a replicative helicase

Origin Remodeling and Opening in Bacteria Rely on Distinct Assembly States of the DnaA Initiator

On helicases and other motor proteins

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