Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair

Wörtz, Julia; Smith, Victoria; Fallmann, Jörg; König, S.; Thuraisingam, Tharani; Walther, Paul; Urlaub, Henning; Stadler, Peter F.; Allers, Thorsten; Hille, Frank; Marchfelder, Anita

doi:10.3389/fmicb.2022.822304

Cited by 4 publications

(6 citation statements)

References 47 publications

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“…Moreover, the contrast between the resistance of the Δcas3 mutant and the sensitivity of the Δcas genes mutant to DNA damaging agents, suggests that different cas genes might affect the susceptibility to DNA damage differently and in opposing directions in H. volcanii . Indeed, Cas1 contributes to DNA repair in E. coli (Babu et al 2011 ), has been recently shown to contribute to DNA repair in H. volcanii , and its deletion alongside the other cas genes could increase sensitivity to DNA damage (Wörtz et al 2022 ).…”

Section: Resultsmentioning

confidence: 99%

“…The three stages of the CRISPR-Cas immunity process (adaptation, maturation, and interference) have been well studied and characterized, and the function of the cas genes has been elucidated in several types and subtypes, including I-B systems, such as that of the haloarchaeon H. volcanii (Maier et al 2015 ). Recent work has shown that at least one protein, namely Cas1, also participates in DNA repair (Babu et al 2011 ; Wörtz et al 2022 ). However, the role of other cas genes in DNA repair is still unknown.…”

Section: Discussionmentioning

confidence: 99%

“…This implies that interactions between archaeal I-B systems and DNA repair are not just possible but rather probable. Indeed, recent work in H. volcanii has shown that the Cas1 integrase, whose primarily role is to integrate new spacers into the CRISPR array, is important for cellular survival of DNA damage caused by oxidative stress and that it can substitute for the DNA repair enzyme Flap endonuclease 1 (Fen1) in cleaving branched intermediate structures (flaps) (Wörtz et al 2022 ). Given that such flap structures are also formed during MMEJ and our observations that Cas3 is involved in the MMEJ process, it is likely that both Cas1 and Cas3 can independently be found at repair sites.…”

Section: Discussionmentioning

confidence: 99%

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An archaeal Cas3 protein facilitates rapid recovery from DNA damage

et al. 2023

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CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type (WT) strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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An archaeal Cas3 protein facilitates rapid recovery from DNA damage

et al. 2023

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“…MONSDA has been successfully applied to a variety of projects, including standard RNA-SEQ and DE analysis [37], Differential transcript usage analysis [38], custom tailored RNA-Seq approaches [29] and a series of projects under review or ongoing. We provide a companion repository https://github.com/jfallmann/MONSDA_companion which will host all configuration files used for analysis of published data and is open to be used as sharing platform all users.…”

Section: Resultsmentioning

confidence: 99%

MONSDA: Modular Organizer of Nextflow and Snakemake driven HTS Data Analysis

Fallmann¹,

Goldmann²,

Stadler³

2022

Preprint

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MONSDA runs HTS data analysis from pre- to postprocessing based on a single configuration file. It wraps Snakemake or Nextflow to run workflow steps involving QC, trimming, mapping, deduplication and differential analysis as well as a set of specific workflows for the generation of genome browser tracks and more. Users profit from many of the advantages of two of the most popular workflow management systems without having to learn all the specifics of Snakemake or Nextflow and can interchange and configure tools according to their specific needs. MONSDA is available via Bioconda, pip and https://github.com/jfallmann/MONSDA

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“…Cas1 and Cas2 are responsible for acquiring new spacers and are the most conserved Cas proteins [ 7 ]. Cas1 exhibits nuclease activity against singe-stranded, double-stranded, and branched DNAs, including Holliday junctions, replication forks and 5′-flaps [ 8 , 9 , 10 ]. The Cas1-Cas2 integrase complex directly catalyses nucleophilic attack of the protospacer at the leader end of the CRISPR array [ 11 , 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus

Liu

Wang

et al. 2022

IJMS

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CRISPR-Cas systems empower prokaryotes with adaptive immunity against invasive mobile genetic elements. At the first step of CRISPR immunity adaptation, short DNA fragments from the invaders are integrated into CRISPR arrays at the leader-proximal end. To date, the mechanism of recognition of the leader-proximal end remains largely unknown. Here, in the Sulfolobus islandicus subtype I-A system, we show that mutations destroying the proximal region reduce CRISPR adaptation in vivo. We identify that a stem-loop structure is present on the leader-proximal end, and we demonstrate that Cas1 preferentially binds the stem-loop structure in vitro. Moreover, we demonstrate that the integrase activity of Cas1 is modulated by interacting with a CRISPR-associated factor Csa3a. When translocated to the CRISPR array, the Csa3a-Cas1 complex is separated by Csa3a binding to the leader-distal motif and Cas1 binding to the leader-proximal end. Mutation at the leader-distal motif reduces CRISPR adaptation efficiency, further confirming the in vivo function of leader-distal motif. Together, our results suggest a general model for binding of Cas1 protein to a leader motif and modulation of integrase activity by an accessory factor.

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Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair

Cited by 4 publications

References 47 publications

An archaeal Cas3 protein facilitates rapid recovery from DNA damage

An archaeal Cas3 protein facilitates rapid recovery from DNA damage

MONSDA: Modular Organizer of Nextflow and Snakemake driven HTS Data Analysis

DNA Motifs and an Accessory CRISPR Factor Determine Cas1 Binding and Integration Activity in Sulfolobus islandicus

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