Identification of a novel nuclease activity in human DDX49 helicase.

Human DDX49 is an emerging target in cancer progression and retroviral diseases through its essential roles in nucleolar RNA processing. Here, we identify nuclease activity of human DDX49, which requires active site aspartate residues within a conserved region of metazoan DDX49s that is absent from yeast and archaeal DDX49 homologues. We provide evidence that DDX49 nuclease activity is facilitated by its helicase activity.

POLD3 as Controller of Replicative DNA Repair.

Multiple modes of DNA repair need DNA synthesis by DNA polymerase enzymes. The eukaryotic B-family DNA polymerase complexes delta (Polδ) and zeta (Polζ) help to repair DNA strand breaks when primed by homologous recombination or single-strand DNA annealing. DNA synthesis by Polδ and Polζ is mutagenic, but is needed for the survival of cells in the presence of DNA strand breaks.

Repurposing Proximity-Dependent Protein Labeling (BioID2) for Protein Interaction Mapping in E. coli.

Methods that identify protein-protein interactions are essential for understanding molecular mechanisms controlling biological systems. Proximity-dependent labeling has proven to be a valuable method for revealing protein-protein interaction networks in living cells. A mutant form of the biotin protein ligase enzyme from Aquifex aeolicus (BioID2) underpins this methodology by producing biotin that is attached to proteins that enter proximity to it.

Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase.

DNA glycosylases protect genetic fidelity during DNA replication by removing potentially mutagenic chemically damaged DNA bases. Bacterial Lhr proteins are well-characterized DNA repair helicases that are fused to additional 600-700 amino acids of unknown function, but with structural homology to SecB chaperones and AlkZ DNA glycosylases. Here, we identify that Escherichia coli Lhr is a uracil-DNA glycosylase (UDG) that depends on an active site aspartic acid residue.

Archaeal Hel308 suppresses recombination through a catalytic switch that controls DNA annealing.

Hel308 helicases promote genome stability in archaea and are conserved in metazoans, where they are known as HELQ. Their helicase mechanism is well characterised, but it is unclear how they specifically contribute to genome stability in archaea. We show here that a highly conserved motif of Hel308/HELQ helicases (motif IVa, F/YHHAGL) modulates both DNA unwinding and a newly identified strand annealing function of archaeal Hel308.

CRISPR-Cas adaptation in Escherichia coli.

Prokaryotes use the adaptive immunity mediated via the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR associated (CRISPR-Cas) system for protection against invading elements such as phages and plasmids. The immunity is achieved by capturing small DNA fragments or spacers from foreign nucleic acids (protospacers) and integrating them into the host CRISPR locus. This step of CRISPR-Cas immunity called 'naïve CRISPR adaptation' requires the conserved Cas1-Cas2 complex and is often supported by variable host proteins that assist in spacer processing and integration.

Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing.

DNA strand breaks are repaired by DNA synthesis from an exposed DNA end paired with a homologous DNA template. DNA polymerase delta (Pol δ) catalyses DNA synthesis in multiple eukaryotic DNA break repair pathways but triggers genome instability unless its activity is restrained. We show that human HelQ halts DNA synthesis by isolated Pol δ and Pol δ-PCNA-RPA holoenzyme.

The HelQ human DNA repair helicase utilizes a PWI-like domain for DNA loading through interaction with RPA, triggering DNA unwinding by the HelQ helicase core.

Genome instability is a characteristic enabling factor for carcinogenesis. HelQ helicase is a component of human DNA maintenance systems that prevent or reverse genome instability arising during DNA replication. Here, we provide details of the molecular mechanisms that underpin HelQ function-its recruitment onto ssDNA through interaction with replication protein A (RPA), and subsequent translocation of HelQ along ssDNA.

A Tryptophan 'Gate' in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity.

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism.

Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9.

CRISPR systems build adaptive immunity against mobile genetic elements by DNA capture and integration catalysed by Cas1-Cas2 protein complexes. Recent studies suggested that CRISPR repeats and adaptation module originated from a novel type of DNA transposons called casposons. Casposons encode a Cas1 homologue called casposase that alone integrates into target molecules single and double-stranded DNA containing terminal inverted repeats (TIRs) from casposons.

Mechanistic insights into Lhr helicase function in DNA repair.

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures.

A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences.

Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for 'gold standard' complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) 'bookmark' sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele.

Cas3 Protein-A Review of a Multi-Tasking Machine.

Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and highlight recent attempts at using Cas3 for genetic editing. We then spotlight involvement of Cas3 in other aspects of cell biology, for which understanding is lacking-these focus on CRISPR systems as regulators of cellular processes in addition to defense against mobile genetic elements.

Adaptation processes that build CRISPR immunity: creative destruction, updated.

Prokaryotes can defend themselves against invading mobile genetic elements (MGEs) by acquiring immune memory against them. The memory is a DNA database located at specific chromosomal sites called CRISPRs (clustered regularly interspaced short palindromic repeats) that store fragments of MGE DNA. These are utilised to target and destroy returning MGEs, preventing re-infection.

CRISPR-Cas immunity, DNA repair and genome stability.

Co-opting of CRISPR-Cas 'Interference' reactions for editing the genomes of eukaryotic and prokaryotic cells has highlighted crucial support roles for DNA repair systems that strive to maintain genome stability. As front-runners in genome editing that targets DNA, the class 2 CRISPR-Cas enzymes Cas9 and Cas12a rely on repair of DNA double-strand breaks (DDSBs) by host DNA repair enzymes, using mechanisms that vary in how well they are understood. Data are emerging about the identities of DNA repair enzymes that support genome editing in human cells.

CRISPR-Cas adaptation in Escherichia coli requires RecBCD helicase but not nuclease activity, is independent of homologous recombination, and is antagonized by 5' ssDNA exonucleases.

Prokaryotic adaptive immunity is established against mobile genetic elements (MGEs) by 'naïve adaptation' when DNA fragments from a newly encountered MGE are integrated into CRISPR-Cas systems. In Escherichia coli, DNA integration catalyzed by Cas1-Cas2 integrase is well understood in mechanistic and structural detail but much less is known about events prior to integration that generate DNA for capture by Cas1-Cas2. Naïve adaptation in E.

DNA replication roadblocks caused by Cascade interference complexes are alleviated by RecG DNA repair helicase.

Cascade complexes underpin E. coli CRISPR-Cas immunity systems by stimulating 'adaptation' reactions that update immunity and by initiating 'interference' reactions that destroy invader DNA. Recognition of invader DNA in Cascade catalysed R-loops provokes DNA capture and its subsequent integration into CRISPR loci by Cas1 and Cas2.

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious.

CRISPR-Cas adaptive immunity and the three Rs.

In this summary, we focus on fundamental biology of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-Cas (CRISPR-associated proteins) adaptive immunity in bacteria. Emphasis is placed on emerging information about functional interplay between Cas proteins and proteins that remodel DNA during homologous recombination (HR), DNA replication or DNA repair. We highlight how replication forks may act as 'trigger points' for CRISPR adaptation events, and the potential for cascade-interference complexes to act as precise roadblocks in DNA replication by an invader MGE (mobile genetic element), without the need for DNA double-strand breaks.

Remodeling and Control of Homologous Recombination by DNA Helicases and Translocases that Target Recombinases and Synapsis.

Recombinase enzymes catalyse invasion of single-stranded DNA (ssDNA) into homologous duplex DNA forming "Displacement loops" (D-loops), a process called synapsis. This triggers homologous recombination (HR), which can follow several possible paths to underpin DNA repair and restart of blocked and collapsed DNA replication forks. Therefore, synapsis can be a checkpoint for controlling whether or not, how far, and by which pathway, HR proceeds to overcome an obstacle or break in a replication fork.

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS.

Background: CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by" an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called "protospacers".

Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair.

Using the ASKA (A Complete Set of Escherichia coli K-12 ORF Archive) library for genome-wide screening of E. coli proteins we identified that expression of ygaQ and rpmG promotes mitomycin C resistance (MMC(R)). YgaQ mediated MMC(R) was independent of homologous recombination involving RecA or RuvABC, but required UvrD.

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').