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Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase (2023)
Journal Article
Buckley, R. J., Lou-Hing, A., Hanson, K. M., Ahmed, N. R., Cooper, C. D., & Bolt, E. L. (2023). Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase. Molecular Microbiology, 120(2), 298-306. https://doi.org/10.1111/mmi.15123

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 un... Read More about Escherichia coli DNA repair helicase Lhr is also a uracil-DNA glycosylase.

Escherichia coli DNA repair helicase Lhr is also a uracil‐DNA glycosylase (2023)
Journal Article
Buckley, R. J., Lou‐Hing, A., Hanson, K. M., Ahmed, N. R., Cooper, C. D. O., & Bolt, E. L. (2023). Escherichia coli DNA repair helicase Lhr is also a uracil‐DNA glycosylase. Molecular Microbiology, 120(2), 298-306. https://doi.org/10.1111/mmi.15123

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 un... Read More about Escherichia coli DNA repair helicase Lhr is also a uracil‐DNA glycosylase.

Archaeal Hel308 suppresses recombination through a catalytic switch that controls DNA annealing (2023)
Journal Article
Lever, R., Simmons, E., Gamble-Milner, R., Buckley, R., Harrison, C., Parkes, A., …Allers, T. (2023). Archaeal Hel308 suppresses recombination through a catalytic switch that controls DNA annealing. Nucleic Acids Research, https://doi.org/10.1093/nar/gkad572

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 s... Read More about Archaeal Hel308 suppresses recombination through a catalytic switch that controls DNA annealing.

Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation (2023)
Journal Article
Killelea, T., Dimude, J. U., He, L., Stewart, A. L., Kemm, F. E., Radovčí, M., …Bolt, E. L. (2023). Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation. Nucleic Acids Research, 51(13), 6914-6926. https://doi.org/10.1093/nar/gkad473

Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on naive adaptation-Cas1-Cas2 targeting of MGE DNA without the aid... Read More about Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation.

CRISPR-Cas adaptation in Escherichia coli (2023)
Journal Article
Mitic, D., Bolt, E. L., & Ivancic-Bace, I. (2023). CRISPR-Cas adaptation in Escherichia coli. Bioscience Reports, 43(3), Article BSR20221198. https://doi.org/10.1042/BSR20221198

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... Read More about CRISPR-Cas adaptation in Escherichia coli.

Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing (2023)
Journal Article
He, L., Lever, R., Cubbon, A., Tehseen, M., Jenkins, T., Nottingham, A. O., …Bolt, E. L. (2023). Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing. Nucleic Acids Research, 51(4), 1740-1749. https://doi.org/10.1093/nar/gkad032

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

A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity (2021)
Journal Article
He, L., Matošević, Z. J., Mitić, D., Markulin, D., Killelea, T., Matković, M., …Bolt, E. L. (2021). A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity. International Journal of Molecular Sciences, 22(6), Article 2848. https://doi.org/10.3390/ijms22062848

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... Read More about A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity.

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 (2021)
Journal Article
Jenkins, T., Northall, S. J., Ptchelkine, D., Lever, R., Cubbon, A., Betts, H., …Bolt, E. L. (2021). 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. NAR Cancer, 3(1), Article zcaa043. https://doi.org/10.1093/narcan/zcaa043

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 molecu... Read More about 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.

Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9 (2021)
Journal Article
Lau, C. H., & Bolt, E. L. (2021). Integration of diverse DNA substrates by a casposase can be targeted to R-loops in vitro by its fusion to Cas9. Bioscience Reports, 41(1), Article BSR20203595. https://doi.org/10.1042/bsr20203595

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

Mechanistic insights into Lhr helicase function in DNA repair (2020)
Journal Article
Buckley, R., Kramm, K., Cooper, C. D. O., Grohmann, D., & Bolt, E. L. (2020). Mechanistic insights into Lhr helicase function in DNA repair. Biochemical Journal, 477(16), 2935–2947. https://doi.org/10.1042/bcj20200379

The DNA helicase 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 pr... Read More about Mechanistic insights into Lhr helicase function in DNA repair.

A gold standard, CRISPR/Cas9-based complementation strategy reliant on 24 nucleotide bookmark sequences (2020)
Journal Article
Seys, F. M., Rowe, P., Bolt, E. L., Humphreys, C. M., & Minton, N. P. (2020). A gold standard, CRISPR/Cas9-based complementation strategy reliant on 24 nucleotide bookmark sequences. Genes, 11(4), Article 458. https://doi.org/10.3390/genes11040458

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. 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 i... Read More about A gold standard, CRISPR/Cas9-based complementation strategy reliant on 24 nucleotide bookmark sequences.

Cas3 Protein—A Review of a Multi-Tasking Machine (2020)
Journal Article
He, L., St. John James, M., Radovcic, M., Ivancic-Bace, I., & Bolt, E. L. (2020). Cas3 Protein—A Review of a Multi-Tasking Machine. Genes, 11(2), Article 208. https://doi.org/10.3390/genes11020208

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... Read More about Cas3 Protein—A Review of a Multi-Tasking Machine.

Adaptation processes that build CRISPR immunity: creative destruction, updated (2019)
Journal Article
Lau, C., Reeves, R., & Bolt, E. (2019). Adaptation processes that build CRISPR immunity: creative destruction, updated. Essays in Biochemistry, 63(2), 227-235. https://doi.org/10.1042/ebc20180073

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 palind... Read More about Adaptation processes that build CRISPR immunity: creative destruction, updated.

CRISPR-Cas immunity, DNA repair and genome stability (2018)
Journal Article
Cubbon, A., Ivancic-Bace, I., & Bolt, E. (2018). CRISPR-Cas immunity, DNA repair and genome stability. Bioscience Reports, 38(5), 1-10. https://doi.org/10.1042/bsr20180457

© 2018 The Author(s). 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... Read More about CRISPR-Cas immunity, DNA repair and genome stability.

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 (2018)
Journal Article
Radovčić, M., Killelea, T., Savitskaya, E., Wettstein, L., Bolt, E. L., & Ivančić-Baće, I. (2018). 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. Nucleic Acids Research, 46(19), 10173-10183. https://doi.org/10.1093/nar/gky799

© The Author(s) 2018. 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 integrati... Read More about 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.

DNA replication roadblocks caused by Cascade interference complexes are alleviated by RecG DNA repair helicase (2018)
Journal Article
Howard, J. L., Killelea, T., Hawkins, M., Howard, J., McGlynn, P., & Bolt, E. L. (2019). DNA replication roadblocks caused by Cascade interference complexes are alleviated by RecG DNA repair helicase. RNA Biology, 16(4), 543-548. https://doi.org/10.1080/15476286.2018.1496773

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 pro... Read More about DNA replication roadblocks caused by Cascade interference complexes are alleviated by RecG DNA repair helicase.

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain (2017)
Journal Article
Northall, S. J., Buckley, R., Jones, N., Penedo, J. C., Soultanas, P., & Bolt, E. L. (2017). DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain. DNA Repair, 57, https://doi.org/10.1016/j.dnarep.2017.07.005

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

Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS (2016)
Journal Article
Majsec, K., Bolt, E. L., & Ivan?i?-Ba?e, I. (in press). Cas3 is a limiting factor for CRISPR-Cas immunity in Escherichia coli cells lacking H-NS. BMC Microbiology, 16(28), https://doi.org/10.1186/s12866-016-0643-5

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

Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair (2015)
Journal Article
Bolt, E. L., Jenkins, T., Russo, V. M., Ahmed, S., Cavey, J., & Cass, S. (2015). Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair. Bioscience Reports, https://doi.org/10.1042/BSR20150249

Using the ASKA (A Complete Set of E. coli K-12 ORF Archive) library for genome-wide screening of E. coli proteins we identified that expression of ygaQ and rpmG promotemitomycin C resistance (MMCR). YgaQ mediated MMCR was independent of homolog... Read More about Identification of Escherichia coli ygaQ and rpmG as novel mitomycin C resistance factors implicated in DNA repair.