An archaeal Cas3 protein facilitates rapid recovery from DNA damage

Abstract

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 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.

Keywords: CRISPR; Cas3; DNA-repair; archaea; haloferax; recombination.

Figure 1.

Δcas3 mutant shows higher resistance to DNA damage. WT (H26), Δcas3 (UG610), and Δcas4 (UG611) cultures were exposed to DNA damage by UV or MMS treatment and plated on Hv-YPC agar. (A) Photographs were taken after 5–10 days (red arrows mark the surviving colonies). (B) Surviving colonies following different UV and MMS doses were enumerated, and the fraction of surviving cells for each strain was calculated. The mean and SE of at least six experiments are shown. (C) Fraction of surviving cells following 0.7 mg/ml MMS treatment. The mean and SE of nine experiments are shown. * = P-value <.05; ** = P-value <.01, Mann–Whitney test, compared to WT.

Figure 2.

Cas3 expression promotes rapid recovery from DNA damage. (A) Growth curves of WT (H26) and Δcas3 (UG610) following treatment with 0.7 mg/ml of MMS. The mean and SE of three biological replicates are shown for each time point. (B) Recovery assay. Colonies were enumerated after 5–10 days of incubation at different time points, before and after 1 h of incubation with 0.1 mg/ml MMS (prerecovery), and after 1 to 3 h of MMS removal (post-recovery). The mean and SE of three experiments are shown.

Figure 3.

Cas3 promotes rapid repair of MMS lesions. (A) MMS lesions quantification following a 1-h MMS (0.1 mg/ml) treatment. The mean and SE of four biological replicates are shown. (B) Percentage of repair after 1-h MMS (0.1 mg/ml) treatment and 1 h of recovery time. The mean and SE of four biological replicates are shown. P = 0.0571 Mann–Whitney test, compared to WT.

Figure 4.

Cas3 helicase activity is responsible for DNA damage sensitivity. WT (H26), Δcas3 (UG610), Δcas3:: cas3 (UG617), Δcas3:: cas3 helicase-dead (UG618), and Δcas3:: cas3 nuclease-dead (UG645) were grown in Hv-YPC (containing 2 mM tryptophan for promoter activation) or in Hv-CA (noninduced medium) and exposed to DNA damage by incubation with 0.7 mg/ml MMS and plated on Hv-YPC. Surviving colonies were enumerated after 5–10 days of incubation and fraction of surviving cells for each mutant was normalized to the WT strain. In each case, the mean and SE of nine experiments are shown. *** = P-value <.001, Mann–Whitney test, compared to WT.

Figure 5.

Δcas3 shows an epistatic (nonadditive) effect in the background of Δmre11- Δrad50. WT (H115), Δmre11 (H203), Δmre11Δcas3 (UG669), Δrad50 (H202), Δrad50Δcas3 (UG670), Δmre11Δrad50 (H204), and Δmre11Δrad50Δcas3 (UG671) cells were exposed to DNA damage by 0.7 mg/ml MMS treatment and plated on Hv-YPC. Surviving colonies were counted after 5–10 days of incubation and the fraction of survival of each mutant was normalized to the WT strain. In each case, the mean and SE of nine experiments are shown. *** = P-value <.001, Mann–Whitney test, compared to WT.

Figure 6.

Pop-in / Recombination efficiencies of cas3 mutants. Integrative plasmid (pTA131-metX volcanii) was transformed into WT (H26), Δcas3 (UG610), cas3 helicase-dead (UG750), and cas3 nuclease-dead (UG767). (A) Pop-in transformation efficiency was calculated as the number of CFU produced by successfully transformed cells with the integrative plasmid divided by the total cells in at least four biological replicates. (B) Normalized recombination efficiency values were obtained by dividing the transformation efficiency obtained with the integrative plasmid (pTA131-metX-volcanii) divided by the transformation efficiency with a replicating plasmid (pTA927) in at least four biological replicates. ** = P-value <.01, Mann–Whitney test, compared to WT.