A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

Fig. 1

E. coli CRISPR system and nuclease activity of YgbT. (A) CRISPR-Cas system of E. coli K12 W3110. Repeats are shown as yellow diamonds, and spacers as colored rectangular boxes. (B, C, D, E) Cleavage of ssDNA (B), ssRNA (C), dsDNA (D) or dsRNA (E) by YgbT (denaturing PAGE/autoradiography). The 5′-[³²P]-labeled ssDNA (34 nt), ssRNA (37 nt) or in a complex with complementary oligonucleotides was incubated without protein (C), with YgbT for the indicated times (37 oC) or in the presence of 10 mM EDTA for 60 min (EDTA). (F) Cleavage of HJ1 by YgbT (native PAGE/autoradiography). The 5′-[³²P]-labeled (*) HJ1 substrate was incubated at 37 oC for 45 min without protein (C) or with YgbT (1) or RuvC (2). (G) Dependence of HJ1 cleavage on YgbT concentration (native PAGE). The 5′-[³²P]-labeled HJ1 substrate (20 nM) was incubated in the absence (1) or in the presence of YgbT (Lanes 2–8: 0.05, 0.1, 0.2, 0.4, 0.8, or 1.0 μg) at 37 oC for 30 min. (H) Effect of divalent metal cations on HJ1 cleavage by YgbT (30 min, 37 °C, 5 mM metals or 10 mM EDTA; native PAGE). (I, J) Denaturing gel analysis of the cleavage of HJ1 by YgbT (triangles) or RuvC (arrows). The HJ1 substrate labeled by [³²P] on different strands (A, B, C, or D) was incubated without protein (C) or with YgbT (1) or RuvC (2). (K) Re-ligation assay of the HJ cleavage products generated by YgbT or RuvC. The [³²P]-labeled asymmetric HJ2 was incubated (30 min at 37oC) without protein (C) or with RuvC or YgbT, and the reaction products were then incubated with T4 DNA ligase prior to PAGE. Re-ligation products are indicated by arrows.

Fig. 2

Nuclease activity of YgbT against branched DNA substrates containing sequences of the E. coli CRISPR. (A) Cleavage of branched DNA substrates by YgbT (native PAGE). The substrates including static HJ (HJ3), replication fork (RF), 5′-flap (5/F), 3′-flap (3/F), and splayed arm duplex (SA) were 5′-[³²P]-labeled on the indicated strand (*) and incubated (30 min at 37 oC) in the absence or in the presence of 0.2 or 0.4 μg of YgbT at pH 7.0 or pH 8.5. (B) Gel-shift assay showing the binding of YgbT to 5′-flap or HJ3 (native PAGE). The 5′-[³²P]-labeled substrates were pre-incubated for 15 min at 25 oC without YgbT (1) or with YgbT (Lanes 2–5: 0.2 μg, 0.4 μg, 0.6 μg, and 0.8 μg). Arrows indicate the position of the DNA-YgbT complexes. (C, D) Denaturing PAGE analysis of YgbT cleavage sites. The 5′-[³²P]-labeled (*) substrates were incubated without or with YgbT (30 min, 37 oC). Panel D shows the sequences of the labeled strands and major cleavage sites of YgbT (arrowheads). The sequence of the E. coli CRISPR repeat-1 is shown in bold letters. (E, F) Cleavage of the CRISPR cruciform-like substrate CF1 by YgbT (denaturing PAGE analysis). The 5′-labeled CF1 (shown in panel F) was incubated with YgbT (0.2 μg) at 37 oC. (G) Alanine replacement mutagenesis of YgbT: cleavage of 5′-flap and HJ3 substrates by purified mutant proteins (30 min at 37 oC; native PAGE/autoradiography).

Fig. 3

Crystal structure of YgbT and the potential active site. (A) Overall structure of the YgbT dimer. (B) Two views of the YgbT monomer (related by a 180° rotation) showing the presence of two domains: the N-terminal β-sandwich-like domain (light-blue) and the C-terminal all-α domain (dark-blue). The position of the potential active site is indicated by the side chains of three conserved residues (shown as sticks). (C) Surface charge presentation of the YgbT monomer showing the presence of several large basic patches representing potential DNA-binding sites (colored in blue). (D) Close-up view of the main basic patch of YgbT located close to the potential active site (E141, H208, and D221) and showing the position of several conserved residues. (E) Superposition of the ssDNA fragment from the E. coli topoisomerase III DNA-binding site (1i7d) onto the potential DNA-binding site of YgbT. The DNA docking was performed using the Surface Screen analysis (Binkowski and Joachimiak, 2008). (F) Close-up stereo view of the YgbT active site from the structure of the C-terminal domain showing the position of three bound sulfate molecules shown as sticks and labelled (1, 2, and 3). The YgbT residues are shown as green sticks along a YgbT ribbon (grey). Two arrows in A and B indicate the protein side and position of the active site shown in C.

Fig. 4

Physical interactions of YgbT with other E. coli proteins. (A) Interactions of YgbT with DNA repair/recombination and Cascade proteins validated by reciprocal SPA-tagging and purification. Yellow nodes represent tagged proteins used as “baits”, and brown edges represent protein-protein associations. (B) Purified YgcH (Cse3) inhibits the HJ cleavage activity of YgbT (native gel/autoradiography). The 5′-[³²P]-labeled HJ3 substrate was pre-incubated for 2 min at room temperature with various amounts of YgcH (0, 0.1, 0.2, 0.3 and 0.4 μg) before the incubation with YgbT (0.3 μg, 45 min, 37 oC). The band-I represents the HJ3 substrate, the band-II - the HJ3 cleavage product (by YgbT), and the band-III (on the top) – the protein/DNA aggregates which are unable enter the gel. (C, D) Co-immunoprecipitation of YgbT with RecB, RecC, RuvB, YgcH, and YgcJ. Panel C show immunoblot analysis of the whole cell lysates (WCL) and anti-FLAG immunoprecipitates (IP) from the untagged DY330 strain (Panel C) or from strains expressing the SPA-tagged Fis or YgbT probed using RecB, RecC and RuvB antisera. Panels D show immunoblot analysis of the WCL and IP from the E. coli C41 (DE3) strain expressing a SPA-tagged Fis or YgbT with His₆-tag YgcJ or YgcH probed using anti-His antibody.

Fig. 5

Genetic interactions of ygbT and DNA damage sensitivity of the ygbT deletion strain. (A) Chromosomal positions of ygbT (green arrow), rec (purple arrows), ruv (yellow arrows), and other gene (red arrows) deletions used as donors (marked with Cm^R) or recipients (marked with Kan^R) in eSGA experiments. csdA and rpoS donor deletions (blue arrows) located close to the rec and ygbT genes served as controls for effects of proximity on recombination. The numbers indicate the gene coordinates on the E. coli chromosome (in minutes). OriT is the F origin of transfer in Hfr Cavalli (12.2 minutes). (B) Validation of ygbT-rec and ygbT-ruv synthetic genetic interactions. rec (Panel I), ruv (Panel II), and control (csdA and rpoS, Panel III) Hfr Cavalli donor deletion strains were crossed with the indicated F^- recipient deletion strains (deleted for ruv, ygbT, or various genes flanking ruv and ygbT), followed by selection on plates containing chloramphenicol and kanamycin. (C) Serial cell dilution assay showing growth inhibition of ygbT-ruvABC double mutants and their single mutants by MMC (0.15 μM). Growth is classified as sensitive to MMC (+) or not sensitive compared with wild-type (−). Also shown are the effects of complementing ΔygbT with pBAD vectors expressing wild-type or inactive mutant YgbT. (D) Effects of the indicated doses of UV irradiation on the survival of ygbT-ruvABC double mutants and their single mutants. The values plotted are averages from three independent experiments.

Fig. 6

Cell morphology of the ygbT deletion strain and intracellular localization of YgbT in response to DNA damage. (A) Intracellular localization of RuvA-YFP, RuvB-YFP, RuvC-YFP, YgbT-YFP, RecN-YFP and MutH-YFP fusion proteins in exponentially growing E. coli cells treated with MMC for 120 min. Scale bar, 2 μM. (B) The percentage of E. coli cells with discrete foci of various YFP fusion proteins in their nucleoids after MMC treatment. In each case, 250 different cells from five independent biological replicates (50 cells per replicate) were examined. Error bars indicate standard deviation from mean. (C) Differential interference contrast images of MMC-induced changes of cell morphology in ygbT-ruvABC, ygbT-recN, ygbT-recO, and ygbT-recF double mutants and their respective single mutants in the presence (+) and in the absence (−) of MMC. Scale bar, 2 μM. The average cell length of these cells is shown in Suppl. Fig. 4. (D) E. coli strains with deleted CRISPR clusters exhibit increased sensitivity to MMC. Serial cell dilution assays showing the effects of MMC (0.15 μM) on the growth of E. coli strains with deletions of one or both CRISPR clusters and/or ygbT. Growth is classified as sensitive to MMC (+) or not sensitive (−) in comparison to wild-type.