Unwinding of a DNA replication fork by a hexameric viral helicase.

Hexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged ß-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding.

Structural and functional analysis of the nucleotide and DNA binding activities of the human PIF1 helicase.

Pif1 is a multifunctional helicase and DNA processing enzyme that has roles in genome stability. The enzyme is conserved in eukaryotes and also found in some prokaryotes. The functions of human PIF1 (hPIF1) are also critical for survival of certain tumour cell lines during replication stress, making it an important target for cancer therapy. Crystal structures of hPIF1 presented here explore structural events along the chemical reaction coordinate of ATP hydrolysis at an unprecedented level of detail. The structures for the apo as well as the ground and transition states reveal conformational adjustments in defined protein segments that can trigger larger domain movements required for helicase action. Comparisons with the structures of yeast and bacterial Pif1 reveal a conserved ssDNA binding channel in hPIF1 that we show is critical for single-stranded DNA binding during unwinding, but not the binding of G quadruplex DNA. Mutational analysis suggests that while the ssDNA-binding channel is important for helicase activity, it is not used in DNA annealing. Structural differences, in particular in the DNA strand separation wedge region, highlight significant evolutionary divergence of the human PIF1 protein from bacterial and yeast orthologues.

DNA substrate recognition and processing by the full-length human UPF1 helicase.

UPF1 is a conserved helicase required for nonsense-mediated decay (NMD) regulating mRNA stability in the cytoplasm. Human UPF1 (hUPF1) is also needed for nuclear DNA replication. While loss of NMD is tolerated, loss of hUPF1 induces a DNA damage response and cell cycle arrest. We have analysed nucleic acid (NA) binding and processing by full-length hUPF1. hUPF1 unwinds non-B and B-form DNA and RNA substrates in vitro. Unlike many helicases involved in genome stability no hUPF1 binding to DNA structures stabilized by inter-base-pair hydrogen bonding was observed. Alternatively, hUPF1 binds to single-stranded NAs (ssNA) with apparent affinity increasing with substrate length and with no preference for binding RNA or DNA or purine compared to pyrimidine polynucleotides. However, the data show a pronounced nucleobase bias with a preference for binding poly (U) or d(T) while d(A) polymers bind with low affinity. Although the data indicate that hUPF1 must bind a ssNA segments to initiate unwinding they also raise the possibility that hUPF1 has significantly reduced affinity for ssNA structures with stacked bases. Overall, the NA processing activities of hUPF1 are consistent with its function in mRNA regulation and suggest that roles in DNA replication could also be influenced by base sequence.

Bacteriophage T5 gene D10 encodes a branch-migration protein.

Helicases catalyze the unwinding of double-stranded nucleic acids where structure and phosphate backbone contacts, rather than nucleobase sequence, usually determines substrate specificity. We have expressed and purified a putative helicase encoded by the D10 gene of bacteriophage T5. Here we report that this hitherto uncharacterized protein possesses branch migration and DNA unwinding activity. The initiation of substrate unwinding showed some sequence dependency, while DNA binding and DNA-dependent ATPase activity did not. DNA footprinting and purine-base interference assays demonstrated that D10 engages these substrates with a defined polarity that may be established by protein-nucleobase contacts. Bioinformatic analysis of the nucleotide databases revealed genes predicted to encode proteins related to D10 in archaebacteria, bacteriophages and in viruses known to infect a range of eukaryotic organisms.

Structural basis for DNA strand separation by a hexameric replicative helicase.

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted 'steric exclusion' model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.

Characterization of an unusual bipolar helicase encoded by bacteriophage T5.

Bacteriophage T5 has a 120 kb double-stranded linear DNA genome encoding most of the genes required for its own replication. This lytic bacteriophage has a burst size of ∼500 new phage particles per infected cell, demonstrating that it is able to turn each infected bacterium into a highly efficient DNA manufacturing machine. To begin to understand DNA replication in this prodigious bacteriophage, we have characterized a putative helicase encoded by gene D2. We show that bacteriophage T5 D2 protein is the first viral helicase to be described with bipolar DNA unwinding activities that require the same core catalytic residues for unwinding in either direction. However, unwinding of partially single- and double-stranded DNA test substrates in the 3'-5' direction is more robust and can be distinguished from the 5'-3' activity by a number of features including helicase complex stability, salt sensitivity and the length of single-stranded DNA overhang required for initiation of helicase action. The presence of D2 in an early gene cluster, the identification of a putative helix-turn-helix DNA-binding motif outside the helicase core and homology with known eukaryotic and prokaryotic replication initiators suggest an involvement for this unusual helicase in DNA replication initiation.

A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding.

The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit-subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity.

Pif1 proteins are helicases that in yeast are implicated in the maintenance of genome stability. One activity of Saccharomyces cerevisiae Pif1 is to stabilize DNA sequences that could otherwise form deleterious G4 (G-quadruplex) structures by acting as a G4 resolvase. The present study shows that human Pif1 (hPif1, nuclear form) is a G4 DNA-binding and resolvase protein and that these activities are properties of the conserved helicase domain (amino acids 206-620 of 641, hPifHD). hPif1 preferentially bound synthetic G4 DNA relative to ssDNA (single-stranded DNA), dsDNA (double-stranded DNA) and a partially single-stranded duplex DNA helicase substrate. G4 DNA unwinding, but not binding, required an extended (>10 nucleotide) 5' ssDNA tail, and in competition assays, G4 DNA was an ineffective suppressor of helicase activity compared with ssDNA. These results suggest a distinction between the determinants of G4 DNA binding and the ssDNA interactions required for helicase action and that hPif1 may act on G4 substrates by binding alone or as a resolvase. Human Pif1 could therefore have a role in processing G4 structures that arise in the single-stranded nucleic acid intermediates formed during DNA replication and gene expression.

Human Pif1 helicase unwinds synthetic DNA structures resembling stalled DNA replication forks.

Pif-1 proteins are 5'-->3' superfamily 1 (SF1) helicases that in yeast have roles in the maintenance of mitochondrial and nuclear genome stability. The functions and activities of the human enzyme (hPif1) are unclear, but here we describe its DNA binding and DNA remodeling activities. We demonstrate that hPif1 specifically recognizes and unwinds DNA structures resembling putative stalled replication forks. Notably, the enzyme requires both arms of the replication fork-like structure to initiate efficient unwinding of the putative leading replication strand of such substrates. This DNA structure-specific mode of initiation of unwinding is intrinsic to the conserved core helicase domain (hPifHD) that also possesses a strand annealing activity as has been demonstrated for the RecQ family of helicases. The result of hPif1 helicase action at stalled DNA replication forks would generate free 3' ends and ssDNA that could potentially be used to assist replication restart in conjunction with its strand annealing activity.

A DNA-binding activity in BPV initiator protein E1 required for melting duplex ori DNA but not processive helicase activity initiated on partially single-stranded DNA.

The papillomavirus replication protein E1 assembles on the viral origin of replication (ori) as a series of complexes. It has been proposed that the ori DNA is first melted by a head-to-tail double trimer of E1 that evolves into two hexamers that encircle and unwind DNA bi-directionally. Here the role of a conserved lysine residue in the smaller tier or collar of the E1 helicase domain in ori processing is described. Unlike the residues of the AAA+ domain DNA-binding segments (beta-hairpin and hydrophobic loop; larger tier), this residue functions in the initial melting of duplex ori DNA but not in the processive DNA unwinding of partially single-stranded test substrates. These data therefore define a new DNA-binding related activity in the E1 protein and demonstrate that separate functional elements for DNA melting and helicase activity can be distinguished. New insights into the mechanism of ori melting are elaborated, suggesting the coordinated involvement of rigid and flexible DNA-binding components in E1.

Papillomavirus E1 helicase assembly maintains an asymmetric state in the absence of DNA and nucleotide cofactors.

Concerted, stochastic and sequential mechanisms of action have been proposed for different hexameric AAA+ molecular motors. Here we report the crystal structure of the E1 helicase from bovine papillomavirus, where asymmetric assembly is for the first time observed in the absence of nucleotide cofactors and DNA. Surprisingly, the ATP-binding sites adopt specific conformations linked to positional changes in the DNA-binding hairpins, which follow a wave-like trajectory, as observed previously in the E1/DNA/ADP complex. The protein's assembly thus maintains such an asymmetric state in the absence of DNA and nucleotide cofactors, allowing consideration of the E1 helicase action as the propagation of a conformational wave around the protein ring. The data imply that the wave's propagation within the AAA+ domains is not necessarily coupled with a strictly sequential hydrolysis of ATP. Since a single ATP hydrolysis event would affect the whole hexamer, such events may simply serve to rectify the direction of the wave's motion.

Transcription activator structure reveals redox control of a replication initiation reaction.

Redox changes are one of the factors that influence cell-cycle progression and that control the processes of cellular proliferation, differentiation, senescence and apoptosis. Proteins regulated through redox-sensitive cysteines have been characterized but specific 'sulphydryl switches' in replication proteins remain to be identified. In bovine papillomavirus type-1, DNA replication begins when the viral transcription factor E2 recruits the viral initiator protein E1 to the origin of DNA replication (ori). Here we show that a novel dimerization interface in the E2 transcription activation domain is stabilized by a disulphide bond. Oxidative cross-linking via Cys57 sequesters the interaction surface between E1 and E2, preventing pre-initiation and replication initiation complex formation. Our data demonstrate that as well as a mechanism for regulating DNA binding, redox reactions can control replication by modulating the tertiary structure of critical protein factors using a specific redox sensor.

High-specific-activity probes and a high-resolution in-gel photo cross-linking assay for protein-DNA complexes.

Photochemical cross-linking has been widely employed to identify proteins interacting with specific sites on DNA. Identification of bound proteins usually relies on transfer of a radiolabel from the DNA to the protein by cross-linking. We set out to fine-map a small viral replication preinitiation complex composed of two protein dimers bound to DNA, the bovine papillomavirus E1E2-ori complex. Here we describe a simple method for generating high-specific-activity probes with a phenyl-azide photoactivatible cross-linking group positioned immediately adjacent to a labeled nucleotide. The method is based on the selective destruction of one 5'-phosphorylated strand of a polymerase chain reaction product with lambda exonuclease and reconstitution of the probe with a phosphorothioate-substituted oligonucleotide, an [alpha-(32)P]dNTP, and thermophilic enzymes. We also developed a high-resolution in-gel cross-linking assay to probe defined protein-DNA complexes. With these methods we have obtained structural information for the papillomavirus E1E2-ori preinitiation complex that would otherwise have been hard to obtain. These approaches should be widely applicable to the study of protein-DNA complexes.

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 approximately 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 approximately 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) approximately 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.

Common determinants in DNA melting and helicase-catalysed DNA unwinding by papillomavirus replication protein E1.

E1 and T-antigen of the tumour viruses bovine papillomavirus (BPV-1) and Simian virus 40 (SV40) are the initiator proteins that recognize and melt their respective origins of replication in the initial phase of DNA replication. These proteins then assemble into processive hexameric helicases upon the single-stranded DNA that they create. In T-antigen, a characteristic loop and hairpin structure (the pre-sensor 1beta hairpin, PS1betaH) project into a central cavity generated by protein hexamerization. This channel undergoes large ATP-dependent conformational changes, and the loop/PS1betaH is proposed to form a DNA binding site critical for helicase activity. Here, we show that conserved residues in BPV E1 that probably form a similar loop/hairpin structure are required for helicase activity and also origin (ori) DNA melting. We propose that DNA melting requires the cooperation of the E1 helicase domain (E1HD) and the origin binding domain (OBD) tethered to DNA. One possible mechanism is that with the DNA locked in the loop/PS1betaH DNA binding site, ATP-dependent conformational changes draw the DNA inwards in a twisting motion to promote unwinding.