Dataset Information

Cas1 and Fen1 Display Commutable Functions During Archaeal DNA Damage Repair

ABSTRACT: CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways either by regulating their expression or via direct protein-protein contact or enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but its function in cellular DNA repair pathways has been proposed before. Here, we analysed the capacity of an archaeal Cas1 from Haloferax volcanii to compensate DNA damage induced by oxidative stress and found that a deletion of the cas1 gene led to severe growth defects after stress induction. In addition, our results indicate that Cas1 is directly involved in DNA damage repair as the enzymatically active site of the protein is crucial for growth rescue under oxidative conditions. Based on biochemical cleavage assays, we propose a mechanism in which Cas1 exerts a similar function like the DNA repair protein Fen1 by resolving branched repair intermediate structures. Overall, the present study broadens our understanding of the functional link between CRISPR-Cas immunity and DNA repair by demonstrating that Cas1 and Fen1 display commutable roles during archaeal DNA damage repair.

INSTRUMENT(S): Orbitrap Exploris 480, Q Exactive HF

ORGANISM(S): Haloferax Volcanii Ds2

SUBMITTER: Henning Urlaub

LAB HEAD: Prof. Dr. Henning Urlaub

PROVIDER: PXD029952 | Pride | 2022-05-20

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	J_Woertz_150721_260721_Expl_3_Band_1.raw	Raw
	J_Woertz_150721_260721_Expl_3_Band_2.raw	Raw
	J_Woertz_150721_260721_Expl_3_Band_3.raw	Raw
	J_Woertz_150721_260721_Expl_3_Band_5.raw	Raw
	J_Woertz_150721_260721_Expl_3_Band_6.raw	Raw

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Publications

Cas1 and Fen1 Display Equivalent Functions During Archaeal DNA Repair.

Wörtz Julia J Smith Victoria V Fallmann Jörg J König Sabine S Thuraisingam Tharani T Walther Paul P Urlaub Henning H Stadler Peter F PF Allers Thorsten T Hille Frank F Marchfelder Anita A

Frontiers in microbiology 20220415

CRISPR-Cas constitutes an adaptive prokaryotic defence system against invasive nucleic acids like viruses and plasmids. Beyond their role in immunity, CRISPR-Cas systems have been shown to closely interact with components of cellular DNA repair pathways, either by regulating their expression or via direct protein-protein contact and enzymatic activity. The integrase Cas1 is usually involved in the adaptation phase of CRISPR-Cas immunity but an additional role in cellular DNA repair pathways has ...[more]

PMID: 35495653

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Project description:CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism. 4 Samples: Protein-associated small RNAs