Assessment and Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

Objective: To study various HNH endonucleases from different sources, including CRISPR-Cas9 protein HNH endonuclease regions, for their conserved motifs, metal binding sites and catalytic amino acids, and to propose a plausible mechanism of action for HNH endonucleases using CRISPR-Cas9 Streptococcus pyogenes as the model enzyme.

Design of the study: Multiple sequence analysis (MSA) was studied for homing endonucleases like CRISPR-Cas9 using Clustal Omega. Other biochemical data available on the CRISPR-Cas9 device, Site-directed mutagenesis (SDM) and X-ray crystallographic data, were also analyzed.

Location and Period of Study: School of Biotechnology, University of Madurai Kamaraj, Madurai, India, 2007 to 2013.

Methodology: Bioinformatics, biochemistry, SDM and X-ray crystallographic data were used for study of HNH endonucleases from various organisms, including CRISPR-Cas9 enzymes. For protein sequence analysis of various HNH endonucleases from different sources, the advanced version of Clustal Omega was used. In order to confirm the possible amino acids involved in the active sites and catalysis, the conserved motifs found by the bioinformatics study were further studied with the data already available from biochemical and SDM and X-ray crystallographic studies of this group of enzymes.

Results: Various forms of homing endonucleases from different sources, including CRISPR-Cas9 enzyme HNH endonuclease regions, exhibit different catalytic regions and metal-binding sites. HNH endonuclease domains are also present in the group I and group II introns of DNA polymerase and reverse transcriptase genes, respectively. However, in all studied homing endonucleases, the catalytic amino acid, i.e. the proton acceptor histidine (His), is completely retained. A plausible mechanism of action for HNH endonucleases is proposed from these results, using CRISPR-Cas9 from Streptococcus pyogenes as the model enzyme.In addition, MSA of different homing endonucleases from various species also showed several highly conserved motifs among them. However, only around the active site regions did some of the HNH endonucleases show consensus. Either -DH—-N or -HH—N forms are grouped out of the catalytic amino acids known from them. There are at least two types of sites known for metal binding and they bind to Mg2+ or Zn2+ or both. Enzyme CRISPR-Cas9 from S. Pyogenes belongs to the -DH-based HNH endonucleases and has a metal binding site of the-DxD-type where it may bind to a catalytic Mg2+ ion. One or two invariant Zn binding CxxC/ CxxxC motifs are found in the other HNH enzymes.

Conclusions: The enzymes of CRISPR-Cas9 are found to be -DH-type where metal-binding is likely to include the first D and the second invariant H acts as the acceptor of protons. By interacting with the nucleotide at the catalytic site, the N in-HNH- Cas9 confers specificity. In this interaction, the nucleophile causing catalysis is seen as a metal-bound water molecule. Homing endonucleases can be used as novel DNA binding and cleaving reagents for a range of applications for genome editing and applications for genome editing have already been identified in zinc finger nucleases.

Author (s) Details

Dr. Peramachi Palanivelu
Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625 021, India (Retd.).

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