PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Cas4 Nuclease and Bacterial Immunity
February 2014
Protein-Nucleic Acid Interaction: Inhibition Through Allostery
July 2013
Stabilizing DNA Single Strands
July 2013
AlkB Homologs
August 2012
Methyl maintenance
May 2012
Follow the RNA leader
December 2011
RNA Chaperone NMB1681
July 2011
Seeing HetR
July 2011
Structure from sequence
July 2011
Added benefits
April 2011
Nitrile Reductase QueF
March 2011
Inhibiting factor
February 2011
Tryptophanyl-tRNA Synthetase
February 2011
Regulating nitrogen assimilation
January 2011
Subtle shifts
January 2011
tRNA Isopentenyltransferase MiaA
August 2010
Mre11 Nuclease
May 2010
Seek and destroy 8-oxoguanine
May 2010
Antibiotics and Ribosome Function
March 2010
Pseudouridine Synthase TruA
November 2009
Get3 into the groove
October 2009
Guanine Nucleotide Exchange Factor Vav1 and Rho GTPase Rac1
October 2009
Proofreading RNA
July 2009
Hda and DNA Replication
June 2009
The elusive helicase
April 2009
Poly(A) RNA recognition
January 2009
Scavenger Decapping Enzyme DcpS
November 2008
Bacteriophage Lambda cII Protein
October 2008
RNase T
July 2008
SARS Coronavirus Nonstructural Protein 1
June 2008

Research Themes DNA and RNA

Added benefits

SBKB [doi:10.1038/sbkb.2011.11]
Featured Article - April 2011
Short description: Biochemical, structural and genetic analyses reveal that the E. coli Cas1 protein YgbT belongs to a novel family of nucleases and that, in addition to antiviral immunity, the CRISPR–Cas system functions in DNA repair.

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.

Arianne Heinrichs

References

  1. M. Babu et al. A dual function of the CRISPR–Cas system in bacterial antivirus immunity and DNA repair.
    Mol. Microbiol. 79, 484-502 (2011). doi:10.1111/j.1365-2958.2010.07465.x

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health