Close-up view of the main basic patch (blue) of YgbT located close to the potential active site, showing the position of several conserved residues.
Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA repeats that are widespread in prokaryotes and that, together with CRISPR-associated proteins (Cas), constitute a novel system of adaptive immunity against viruses and plasmids. The Escherichia coli K12 strain W3110 encodes three core Cas proteins and five non-core Cas proteins. Together, these eight proteins form the so-called Cascade complex, which processes long CRISPR RNA transcripts into short RNAs.
To characterize the biochemical activity of the E. coli Cas1 protein YgbT, Babu et al. (PSI MCSG) tested the activity of the purified protein on a range of linear DNA and RNA substrates and found that it has a divalent metal cation–dependent nuclease activity against single-stranded DNAs (ssDNAs), single-stranded RNAs (ssRNAs) and short dounle-stranded DNAs (dsDNAs). Because the resolution of Holliday junctions requires the cleavage of ssDNAs, they tested YgbT for Holliday junction resolvase activity and found that it can cleave Holliday junctions as well as some other branched DNA substrates that represent various intermediates of DNA repair. Its range of nucleic acid substrate specificities also suggests that YgbT might represent a new family of 5′-flap endonucleases.
The crystal structure of the full-length YgbT protein shows that it forms a homodimer. Similar to previously crystallized Cas1 proteins, the YgbT monomer consists of a small N-terminal domain connected by a flexible linker to a larger C-terminal domain, which contains the active site. Surface charge analysis reveals the presence of several large patches of positively charged residues, which represent potential DNA-binding sites. Mutations of four conserved amino acids near the main patch indicate that they are crucial for nuclease activity, suggesting that the active site is located close to the potential DNA-binding site in the C-terminal domain.
The nuclease activity of YgbT against branched DNAs suggests that it might participate in one or more DNA repair–recombination pathways. This idea is supported by several lines of genetic and biochemical evidence. For example, knockout of the ygbT gene results in increased sensitivity to DNA damage caused by mitomycin C (MMC) or UV light, but resistance can be restored by the presence of catalytically active, but not inactive, YgbT. Also, YgbT interacts physically with several key repair proteins, including RecB, RecC and RuvB, as well as two non-core Cas proteins that are part of the Cascade complex. Genetic interaction studies further implicate YgbT in both the recBC and RecF recombinational repair pathways. Bacterial strains showing hypersensitivity to MMC also exhibit impaired cell division and chromosomal segregation in the presence of unrepaired DNA damage. The ygbT deletion strain similarly forms elongated, non-septate, multi-nucleate cells in the presence of MMC, suggesting that YgbT has some role in resolving chromosomes during cell division.
Finally, the ability of YgbT to cleave branched DNA substrates in vitro suggests that this activity might contribute to the addition or removal of CRISP spacers, which is thought to occur through DNA recombination. Indeed, genetic interaction studies suggest that CRISPRs are required for the function of YgbT in DNA repair. However, the molecular mechanism underlying the CRISPR-dependent role of YgbT in DNA repair remains to be elucidated.