The results presented here indicate that the E. coli
Cas1 protein YgbT is a novel nuclease that can cleave HJs and other branched DNA substrates, as well as linear ss/ds DNAs and ssRNAs. To date, E. coli
is known to encode one functional HJ resolvase, RuvC, and one cryptic resolvase, RusA, which is normally not expressed (Benson and West, 1994
; Sharples et al., 1994
). In contrast to RuvC, YgbT shows no apparent target sequence specificity. In addition, YgbT cleaves in vitro
a broad range of branched DNA substrates (asymmetrical Holliday junction, replication fork, 5′-flap, 3′-flap and splayed arm substrates) which represent various intermediates of DNA recombination and repair (). Like human MUS81-EME1 and SLX1, YgbT produces multiple HJ cleavage products and can also cleave replication fork and 3′-flap structures; however, in addition, YgbT is active with 5′-flap and splayed arm substrates which are not cleaved by MUS81 (Constantinou et al., 2002
). The 5′-flap endonucleases, which are required for the removal of RNA primers during replicative and repair DNA synthesis and typically can cleave both RNA and DNA, are encoded as distinct proteins in eukaryotes (FEN-1), archaea and some DNA viruses, whereas their bacterial homologs are N-terminal domains of DNA polymerase I (Shen et al., 1998
). Given that YgbT cleaves both ssRNA and 5′-flap DNAs but is unrelated to the FEN-1 family it might represent a new group of stand-alone flap endonucleases.
When compared to one another, the three experimentally characterized Cas1 proteins display related but different biochemical properties: SSO1450 binds DNA/RNA but appears not to possess nuclease activity, PaCas1
cleaves ss/dsDNA, whereas YgbT is also active against branched DNA substrates and linear ssRNAs. These proteins belong to different CRISPR subtypes (Makarova et al., 2006
) and share rather low overall sequence similarity (18.9 – 21.6 % of sequence identity), suggesting that they might possess genuinely different substrate preferences. This possibility is consistent with the presence of several (2 to 5) distinct paralogous Cas1 proteins in many bacteria and archaea. Further, detailed biochemical studies of Cas1 proteins from diverse organisms are required to delineate the functional versatility of this protein family.
The ability of YgbT to cleave branched DNA substrates in vitro
suggests that this activity might contribute to the addition/removal of CRISPR spacers, which is proposed to proceed through DNA recombination (Mojica et al., 2009
). Four strictly conserved amino acid residues of YgbT (E141, H208, D218 and D221) are critical for activity and represent its active site, which is located close to the potential DNA-binding site in the C-terminal domain. Further detailed analysis of the role of YgbT in the CRISPR mechanism requires the use of a natural experimental model of CRISPR spacer addition/removal, which has yet to be established in E. coli
The incorporation of a novel CRISPR spacer and the accompanying repeat apparently involves the recognition of foreign DNA followed by processing steps and insertion into the CRISPR cluster. Given that YgbT showed no obvious sequence selectivity in the cleavage of branched DNAs, we hypothesize that other Cas (and non-Cas) proteins are also involved in the formation of novel CRISPR spacers, whereas YgbT might contribute to one of the final steps in the integration of the spacers into the chromosomal or plasmid DNA (e.g., HJ resolution or flap substrate cleavage). The physical interaction between YgbT and two components of the Cascade complex (Cse4/CasC and Cse3/CasE) reported in this work also suggests that Cascade might contribute to the integration of new spacers in E. coli.
The key conclusion of the present work is that YgbT and CRISPR are involved in one or more repair-recombination pathways and contribute to the resistance of E. coli
to at least some types of DNA damage and chromosome segregation. This conclusion is consistently supported by several lines of genetic and biochemical evidence: (1) knockout of the ygbT
gene results in a substantial increase in the sensitivity of E. coli
to DNA damage caused by UV or MMC; (2) the rescue of the knockout mutants requires catalytically active YgbT, indicating that the apparent role of YgbT in repair-recombination depends on its demonstrated endonuclease activity towards the characteristic intermediates of several DNA repair pathways, including recombinational (HJs and splayed arms), base excision (5′-flaps), and nucleotide excision (3′-flaps) pathways; (3) YgbT physically interacts with several key repair proteins including RecB, RecC and RuvB; (4) the ygbT
gene genetically interacts with repair genes, including synthetic-sick interactions with recB
, indicative of functional redundancy, and alleviating interactions with ruvABC
, indicative of involvement in the same repair-recombination pathway(s); (5) YgbT is recruited to DSBs in MMC-treated cells; (6) YgbT is required for cell division in MMC-treated cells, as evidenced by the unusual morphology of ygbT
knockouts that resembles the morphology of knockout mutants of other repair genes upon treatment with DNA-damaging agents. In addition, we found that the function of YgbT in DNA repair apparently requires interaction with CRISPRs because deletion of the CRISPRs or the double deletion of the CRISPRs and ygbT
led to the same phenotype as the ygbT
knockout. The specific role of CRISPRs in repair remains to be elucidated, but the involvement of their recombinogenic potential seems plausible; previously, it has been suggested that CRISPRs mediate genome rearrangements in Thermotogales
via recombination (DeBoy et al., 2006
Thus, YgbT appears to be a multifunctional nuclease that can cleave various branched DNA intermediates produced by DNA repair pathways, chromosome segregation mechanisms, and (potentially) by the CRISPR system. More generally, our results suggest an intrinsic connection between the function of the CRISPR-Cas system in the acquired antiviral immunity and its emerging role in DNA repair in prokaryotes. These findings reconcile the latest observations on the antiviral functions of this system that depend on unique spacers homologous to viral genes (Horvath and Barrangou, 2010
; Karginov and Hannon, 2010
) and the earlier hypothesis on a repair function of the Cas proteins (Makarova et al., 2002
) that was proposed before the discovery of the virus-specific spacers, solely on the basis of the predicted enzymatic activities of the Cas proteins (a helicase, a polymerase and multiple nucleases). Furthermore, a dual function of the CRISPR-Cas system in defense and repair is compatible with the typically small fraction of virus-specific CRISPR spacers and the inability of some of these spacers to protect the host against infection (Semenova et al., 2009
; van der Ploeg, 2009
; Zegans et al., 2009
). It is conceivable that the array of diverse enzymes and nucleic acid-binding proteins associated with the CRISPRs evolved at early stages of the evolution of bacteria and archaea within the intrinsically interlinked contexts of DNA damage repair and antiviral defense. Further research into the functions of various CRISPR-Cas systems should reveal the relationships between its apparently diverse functions and more specific roles of the individual components.