Helicase

Telomeres play critical roles in protecting genome stability, and their dysfunction contributes to cancer and age-related degenerative diseases. The precise architecture of telomeres, including their single-stranded 3′ overhangs, bound proteins, and ability to form unusual secondary structures such as t-loops, is central to their function and thus requires careful processing by diverse factors. Furthermore, telomeres provide unique challenges to the DNA replication and recombination machinery, and are particularly suited for extension by the telomerase reverse transcriptase. Helicases use the energy from NTP hydrolysis to track along DNA and disrupt base pairing. Here we review current findings concerning how helicases modulate several aspects of telomere form and function.

DNA polymerase theta (Polq) has been identified as a crucial alternative non-homologous end-joining fac- tor in mammalian cells. Polq is upregulated in a range of cancer cell types defective in homologous recom- bination, and knockdown has been shown to inhibit cell survival in a subset of these, making it an attrac- tive target for cancer treatment. We present crystal structures of the helicase domain of human Polq in the presence and absence of bound nucleotides, and a characterization of its DNA-binding and DNA- stimulated ATPase activities. Comparisons with related helicases from the Hel308 family identify several unique features. Polq exists as a tetramer both in the crystals and in solution. We propose a model for DNA binding to the Polq helicase domain in the context of the Polq tetramer, which suggests a role for the helicase domain in strand annealing of DNA templates for subsequent processing by the polymerase domain.

Helicobacter pylori, an important bacterial pathogen, causes gastric ulcer and gastric adenocarcinoma in humans. The fundamentals of basic biology such as DNA replication are poorly understood in this pathogen. In the present study, we report the cloning and functional characterization of the single-stranded DNA (ssDNA) binding protein from H. pylori. The N-terminal DNA binding domain shows significant homology with E. coli single-stranded DNA binding protein (SSB), whereas the C-terminal domain shows less homology. The overall DNA-binding activity and tetramerization properties, however, remain unaffected. In in vitro experiments with purified proteins, H. pylori (Hp) SSB bound specifically to ssDNA and modulated the enzymatic ATPase and helicase activity of HpDnaB helicase. HpSSB and HpDnaB proteins were co-localized in sharp, distinct foci in exponentially growing H. pylori cells, whereas both were spread over large areas in its dormant coccoid form, suggesting the absence of active replication forks in the latter. These results confirm the multiple roles of SSB during DNA replication and provide evidence for altered replicative metabolism in the spiral and coccoid forms that may be central to the bacterial physiology and pathogenesis.

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3' single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational ...

RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ149–616) shows a prominent b-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the b-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the b-hairpin is required for dimer forma- tion in RECQ149–616 and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ149–616 dimers in solution and demonstrated that dimer formation favours DNA un- winding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein–protein contacts are required for tetramer formation, one involves the b-hairpin and the other the N-terminus of RECQ1, suggesting a non- hierarchical mechanism of tetramer assembly.

Summary RNA silencing in transgenic and virus-infected plants involves a mobile silencing signal that can move cell-to- cell and systemically through the plant. It is thought that this signal can influence long-distance movement of viruses because protein suppressors of silencing encoded in viral genomes are required for long-distance virus movement. However, until now, it was not known whether the mobile

RuvBL1 (RuvB-like 1) and its homolog RuvBL2 are evolutionarily highly conserved AAA(+) ATPases essential for many cellular activities. They play an important role in chromatin remodeling, transcriptional regulation and DNA damage repair. RuvBL1 and RuvBL2 are overexpressed in different types of cancer and interact with major oncogenic factors, such as β-catenin and c-Myc regulating their function. We solved the first three-dimensional crystal structure of the human RuvBL complex with a truncated domain II and show that this complex is competent for helicase activity. The structure reveals a dodecamer consisting of two heterohexameric rings with alternating RuvBL1 and RuvBL2 monomers bound to ADP/ATP, that interact with each other via the retained part of domain II. The dodecameric quaternary structure of the R1ΔDII/R2ΔDII complex observed in the crystal structure was confirmed by small-angle X-ray scattering analysis. Interestingly, truncation of domain II led to a substantial incre...

The kinetic mechanism by which Escherichia coli RecBCD helicase unwinds duplex DNA was studied using a fluorescence stopped-flow method. Single turnover DNA unwinding experiments were performed using a series of fluorescently labeled DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. All or no DNA unwinding time courses were obtained by monitoring the changes in fluorescence resonance energy transfer between a Cy3 donor and Cy5 acceptor fluorescent pair placed on opposite sides of a nick in the duplex DNA. From these experiments one can determine the average rates of DNA unwinding as well as a kinetic step-size, defined as the average number of base-pairs unwound between two successive rate-limiting steps repeated during DNA unwinding. In order to probe how the kinetic step-size might relate to a mechanical step-size, we performed single turnover experiments as a function of [ATP] and temperature. The apparent unwinding rate constant, kUapp, decreases with dec...

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Rvb1 and Rvb2 are highly conserved proteins present in archaea and eukaryotes. These proteins are members of a large superfamily of ATPases associated with diverse cellular activities--the AAA(+) superfamily. The Rvbs have been found in multiprotein complexes that have wide ranges of functions, including DNA repair, transcription, chromatin remodeling, ribosomal RNA processing, and small nucleolar RNA accumulation. Here we show that yeast Rvb1 and Rvb2 form a heterohexameric ring structure rather than the double-hexameric ring structure proposed to be formed by the human proteins. The yeast Rvb1/2 complex has enhanced ATPase activity compared with the individual Rvb proteins; furthermore, the ATPase activity of the Rvb1/2 complex is further increased in the presence of double-stranded DNA with 5' or 3' overhangs. The yeast Rvb1/2 ring undergoes nucleotide-dependent conformational changes as observed by electron microscopy. In addition, consistent with a role for these protei...