Edward L. Bolt

Archaeal Hel308 helicase targets replication forks in vivo and in vitro and unwinds lagging strands

Mutations in mammalian and Drosophila Hel308 and PolQ paralogues cause genome instability but their helicase functions are mysterious. By in vivo and in vitro analysis, we show that Hel308 from archaea (Hel308a) may act at stalled replication forks. Introducing hel308a into Escherichia coli dnaE strains that conditionally accumulate stalled forks caused synthetic lethality, an effect indistinguishable from E.coli RecQ. Further analysis in vivo indicated that the effect of hel308a is exerted independently of homologous recombination. The minimal biochemical properties of Hel308a protein were the same as human Hel308. We describe how helicase actions of Hel308a at fork structures lead specifically to displacement of lagging strands. The invading strand of D-loops is also targeted. Using archaeal Hel308, we propose models of action for the helicase domain of PolQ, promoting loading of the translesion polymerase domain. We speculate that removal of lagging strands at stalled forks by Hel308 promotes the formation of initiation zones, priming restart of lagging strand synthesis.

Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition

The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements. The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus, a process known as Adaptation, which is dependent on the Cas1 and Cas2 proteins. We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates, which represents the reverse- or disintegration reaction. Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity, with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus. Cas2 is not required for this activity and does not influence the specificity. This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process. DOI: http://dx.doi.org/10.7554/eLife.08716.001

Replication fork regression in vitro by the Werner syndrome protein (WRN): Holliday junction formation, the effect of leading arm structure and a potential role for WRN exonuclease activity

The premature aging and cancer-prone disease Werner syndrome stems from loss of WRN protein function. WRN deficiency causes replication abnormalities, sensitivity to certain genotoxic agents, genomic instability and early replicative senescence in primary fibroblasts. As a RecQ helicase family member, WRN is a DNA-dependent ATPase and unwinding enzyme, but also possesses strand annealing and exonuclease activities. RecQ helicases are postulated to participate in pathways responding to replication blockage, pathways possibly initiated by fork regression. In this study, a series of model replication fork substrates were used to examine the fork regression capability of WRN. Our results demonstrate that WRN catalyzes fork regression and Holliday junction formation. This process is an ATP-dependent reaction that is particularly efficient on forks containing single-stranded gaps of at least 11–13 nt on the leading arm at the fork junction. Importantly, WRN exonuclease activity, by digesting the leading daughter strand, enhances regression of forks with smaller gaps on the leading arm, thus creating an optimal structure for regression. Our results suggest that the multiple activities of WRN cooperate to promote replication fork regression. These findings, along with the established cellular consequences of WRN deficiency, strongly support a role for WRN in regression of blocked replication forks.

Molecular insights into DNA interference by CRISPR-associated nuclease-helicase Cas3

Significance Bacteria can repel invader DNA and RNA molecules by using an adaptive immunity mechanism called clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas. CRISPR loci in a host genome are a repository of DNA fragments obtained from previous encounters with an invader, which can be transcribed and activated into short RNA molecules (crRNA) with sequences complementary to invader DNA or RNA. In some CRISPR-Cas systems, crRNA is assembled into a targeting complex called “Cascade” that seeks invader DNA to form an R-loop that triggers recruitment of a nuclease-helicase, Cas3, to destroy invader DNA. In this study, we show atomic resolution structures of a full-length Cas3, revealing how Cas3 coordinates binding, ATP-dependent translocation, and nuclease digestion of invader DNA. Mobile genetic elements in bacteria are neutralized by a system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Type I CRISPR-Cas systems use a “Cascade” ribonucleoprotein complex to guide RNA specifically to complementary sequence in invader double-stranded DNA (dsDNA), a process called “interference.” After target recognition by Cascade, formation of an R-loop triggers recruitment of a Cas3 nuclease-helicase, completing the interference process by destroying the invader dsDNA. To elucidate the molecular mechanism of CRISPR interference, we analyzed crystal structures of Cas3 from the bacterium Thermobaculum terrenum, with and without a bound ATP analog. The structures reveal a histidine-aspartate (HD)-type nuclease domain fused to superfamily-2 (SF2) helicase domains and a distinct C-terminal domain. Binding of ATP analog at the interface of the SF2 helicase RecA-like domains rearranges a motif V with implications for the enzyme mechanism. The HD-nucleolytic site contains two metal ions that are positioned at the end of a proposed nucleic acid-binding tunnel running through the SF2 helicase structure. This structural alignment suggests a mechanism for 3′ to 5′ nucleolytic processing of the displaced strand of invader DNA that is coordinated with ATP-dependent 3′ to 5′ translocation of Cas3 along DNA. In agreement with biochemical studies, the presented Cas3 structures reveal important mechanistic details on the neutralization of genetic invaders by type I CRISPR-Cas systems.

Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity

CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.

The Conjugative DNA Translocase TrwB Is a Structure-specific DNA-binding Protein

TrwB is a DNA-dependent ATPase involved in DNA transport during bacterial conjugation. The protein presents structural similarity to hexameric molecular motors such as F1-ATPase, FtsK, or ring helicases, suggesting that TrwB also operates as a motor, using energy released from ATP hydrolysis to pump single-stranded DNA through its central channel. In this work, we have carried out an extensive analysis with various DNA substrates to determine the preferred substrate for TrwB. Oligonucleotides with G-rich sequences forming G4 DNA structures were the optimal substrates for TrwB ATPase activity. The protein bound with 100-fold higher affinity to G4 DNA than to single-stranded DNA of the same sequence. Moreover, TrwB formed oligomeric protein complexes only with oligonucleotides presenting such a G-quadruplex DNA structure, consistent with stoichiometry of six TrwB monomers to G4 DNA, as demonstrated by gel filtration chromatography and analytical ultracentrifugation experiments. A protein-DNA complex was also formed with unstructured oligonucleotides, but the molecular mass corresponded to one monomer protein bound to one oligonucleotide molecule. Sequences capable of forming G-quadruplex structures are widespread through genomes and are thought to play a biological function in transcriptional regulation. They form stable structures that can obstruct DNA replication, requiring the action of specific helicases to resolve them. Nevertheless, TrwB displayed no G4 DNA unwinding activity. These observations are discussed in terms of a possible role for TrwB in recognizing G-quadruplex structures as loading sites on the DNA.

Molecular biology of Hel308 helicase in archaea

Hel308 is an SF2 (superfamily 2) helicase with clear homologues in metazoans and archaea, but not in fungi or bacteria. Evidence from biochemistry and genetics implicates Hel308 in remodelling compromised replication forks. In the last 4 years, significant advances have been made in understanding the biochemistry of archaeal Hel308, most recently through atomic structures from cren- and eury-archaea. These are good templates for SF2 helicase function more generally, highlighting co-ordinated actions of accessory domains around RecA folds. We review the emerging molecular biology of Hel308, drawing together ideas of how it may contribute to genome stability through the control of recombination, with reference to paradigms developed in bacteria.

RusA proteins from the extreme thermophile Aquifex aeolicus and lactococcal phage r1t resolve Holliday junctions

The RusA protein of Escherichia coli is a DNA structure‐specific endonuclease that resolves Holliday junction intermediates formed during DNA replication, recombination and repair by introducing symmetrically paired incisions 5′ to CC dinucleotides. It is encoded by the defective prophage DLP12, which raises the possibility that it may be of bacteriophage origin. We show that rusA‐like sequences are indeed often associated with prophage sequences in the genomes of several bacterial species. They are also found in many bacteriophages, including Lactococcus lactis phage r1t. However, rusA is also present in the chromosome of the hyperthermophilic bacterium Aquifex aeolicus. In this case, there is no obvious association of rusA with prophage‐like sequences. Given the ancient lineage of Aquifex aeolicus, this observation provides the first indication that RusA may be of bacterial origin. The RusA proteins of A. aeolicus and bacteriophage r1t were purified and shown to resolve Holliday junctions. The r1t enzyme also promotes DNA repair in strains lacking the RuvABC resolvase. Both enzymes cleave junctions in a sequence‐dependent manner, but the A. aeolicus RusA shows a different sequence preference (3′ to TG) from the E. coli protein (5′ to CC), and the r1t RusA has relaxed sequence dependence, requiring only a single cytosine.

RusA Holliday junction resolvase: DNA complex structure—insights into selectivity and specificity

We have determined the structure of a catalytically inactive D70N variant of the Escherichia coli RusA resolvase bound to a duplex DNA substrate that reveals critical protein–DNA interactions and permits a much clearer understanding of the interaction of the enzyme with a Holliday junction (HJ). The RusA enzyme cleaves HJs, the fourway DNA branchpoints formed by homologous recombination, by introducing symmetrical cuts in the phosphodiester backbone in a Mg2+ dependent reaction. Although, RusA shows a high level of selectivity for DNA junctions, preferring to bind fourway junctions over other substrates in vitro, it has also been shown to have appreciable affinity for duplex DNA. However, RusA does not show DNA cleavage activity with duplex substrates. Our structure suggests the possible basis for structural selectivity as well as sources of the sequence specificity observed for DNA cleavage by RusA.

Physical interaction between archaeal DNA repair helicase Hel308 and Replication Protein A (RPA).

Hel308 is a super-family 2 helicase in archaea with homologues in higher eukaryotes (HelQ and PolQ) that contribute to repair of DNA strand crosslinks (ICLs). However, the contribution of Hel308 to repair processes in archaea is far from clear, including how it co-operates with other proteins of DNA replication, repair and recombination. In this study we identified a physical interaction of Hel308 with RPA. Hel308 did not interact with SSB, and interaction with RPA required a conserved amino acid motif at the Hel308 C-terminus. We propose that in archaea RPA acts as a platform for loading of Hel308 onto aberrant single-stranded DNA (ssDNA) that arises at blocked replication forks. In line with data from a human Hel308 homologue, the helicase activity of archaeal Hel308 was only modestly stimulated (1.5-2 fold) by RPA under some conditions, and much less so than for other known interactions between helicases and single strand DNA (ssDNA) binding proteins. This supports a model for RPA localising Hel308 to DNA damage sites in archaea, rather than it directly stimulating the mechanism of helicase unwinding.