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

Main Article Content

Peramachi Palanivelu


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

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

Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007 and 2013.

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

Results: Different types of homing endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 enzymes exhibit different catalytic regions and metal-binding sites. However, the catalytic amino acid, i.e., the proton acceptor histidine (His), is completely conserved in all homing endonucleases analyzed. From these data, a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 from Streptococcus pyogenes, as the model enzyme is proposed. Furthermore, multiple sequence alignment (MSA) of various homing endonucleases from different organisms showed many highly conserved motifs also among them. However, some of the HNH endonucleases showed consensus only around the active site regions. Possible catalytic amino acids identified among them belong to either -DH---N or -HH--N types. There are at least two types of metal-binding sites and bind Mg2+ or Zn2+ or both. The CRISPR-Cas9 enzyme from S. pyogenes belongs to the -DH- based HNH endonucleases and possesses –DxD- type metal-binding site where it possibly binds to a Mg2+ ion. The other HNH enzymes possess one or two invariant Zn binding CxxC/ CxxxC motifs.

Conclusions: The CRISPR-Cas9 enzymes are found to be -DH- type where the first D is likely to involve in metal-binding and the second invariant H acts as the proton acceptor and the N in –HNH- Cas9 confers specificity by interacting with the nucleotide near the catalytic region. In this communication, a metal-bound water molecule is shown as the nucleophile initiating catalysis. Homing endonucleases may be used as novel DNA binding and cleaving reagents for a variety of genome editing applications and Zinc finger nucleases have already found applications in genome editing.

Homing endonucleases, HNH endonucleases, CRISPR-Cas9, Colicins, Pyocins, group II intron reverse transcriptases, CRISPR-Cas9-HNH endonucleases, Conserved motifs, active sites, mechanism of action.

Article Details

How to Cite
Palanivelu, P. (2020). Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases. International Journal of Biochemistry Research & Review, 29(6), 1-25.
Original Research Article


Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli and identification of the gene product. Journal of Bacteriology. 1987;169:5429-5433.

Deveau H, Garneau JE, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Ann Rev Microbiol. 2010;64:475–493.

Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327:167–170.

Marraffini LA, Sontheimer EJ. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet. 2010;11:181–190.

Terns MP, Terns RM. CRISPR-based adaptive immune systems. Curr Opin Microbiology. 2011;14:321–327.

Pandey VK, Tripathi A, Bhushan R, Ali A, et al. Application of CRISPR/Cas9 genome editing in genetic disorders: A systematic review up to date. J Genet Syndr Gene Ther. 2017;8:2.

Selle K, Barrangou R. CRISPR-based technologies and the future of food science. J Food Sci. 2015;80:R2367-R2372.

Jansen R, Embden JDAV, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43:1565-1575.

Mojica FJM, Díez-Villaseñor C, Soria E, Juez G. Biological significance of a family of regularly spaced repeats in the genomes of archaea, bacteria and mitochondria. Mol Microbiol. 2000;36:244-246.

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007;315:1709–1712.

Brouns SJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJ, Snijders AP, Dickman MJ, Makarova KS, Koonin EV, van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008;321:960–964.

Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in Staphylococci by targeting DNA. Science. 2008;322:1843–1845.

Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV. Evolution and classification of the CRISPR–Cas systems. Nat Rev Microbiol. 2011;9:467–477.

Athukoralage JS, Graham S, Grüschow S, Rouillon C, White MF. A type III CRISPR ancillary ribonuclease degrades its cyclic oligoadenylate activator.


Ahn WC, Par KH, Bak IS, Song HN, An Y, Lee SJ, Jung M, Yoo KW, Yu DY, Kim YS, Oh BH, Woo EJ. In vivo genome editing using the Cpf1 ortholog derived from Eubacterium eligens. Nature Scientific Reports. 2019;9:13911.

Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci (USA). 2012;109:E2579–2586.

Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014;156:935-949.

Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013;31:827–832.

Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–1278.

Jurica MS, Stoddard BL. Homing endonucleases: Structure, function and evolution. Cell Mol Life Sci. 1999;55:1304-26.

Ramalingam R, Prasad R, Shivapriya R, Dharmalingam K. Molecular cloning and sequencing of mcrA locus and identification of mcrA protein in Escherichia coli. J Biosci. 1992;17:217–232.

Huang H, Yuan HS. The conserved asparagine in the HNH motif serves an important structural role in metal finger endonucleases. J Mol Biol. 2007;368:812-821.

Wy KU, Liu YW, Hsu YC, Liao CC, Liang PH, Yuan HS, Chak KF. The zinc ion in the HNH motif of the endonuclease domain of colicin E7 is not required for DNA binding but is essential for DNA hydrolysis. Nucleic Acids Res. 2002;30:1670-1678.

Sano Y, Kageyama M. Genetic determinant of Pyocin AP41 as an insert in the Pseudomonas aeruginosa Chromosome. Journal of Bacteriology. 1984;158:562-570.

Shinomiya T, Sano Y, Kikuchi A, Kageyama M. Mapping of pyocin genes on the chromosome of Pseudotonis aeruginosa using plasmid R68.45. In S. Mitsuhashi (Ed.), Drug resistance in bacteria. Japan Scientific Societies Press, Tokyo. 1982;213-217.

Gorbalenya AE. Self-splicing group I and group I1 introns encode homologous (putative) DNA endonucleases of a new family. Protein Science. 1994;3:1117- 1120.

Flick KE, Jurica MS, Monnat RJ Jr, Stoddard BL. DNA binding and cleavage by the nuclear intron-encoded homing endonuclease I-PpoI. Nature. 1998;394: 96-101.

Mannino SJ, Jenkins CL, Raines RT. Chemical mechanism of DNA cleavage by the homing endonuclease I-PpoI. Biochemistry. 1999;38:16178-86.

Saravanan M, Bujnicki JM, Cymerman IA, Rao DN, Nagaraja VN. Type II restriction endonuclease R.KpnI is a member of the HNH nuclease superfamily. Nucleic Acids Res. 2004;32:6129–6135.

Raaijmakers H, Toro I, Birkenbihl R, Kemper B, Suck D. Conformational flexibility in T4 endonuclease VII revealed by crystallography: Implications for substrate binding. J. Mol. Biol. 2001;308: 311–323.

Giraud-Panis MJE, Duckett DR, Lilley DMJ. The modular character of a DNA junction resolving enzyme: A zinc binding motif in T4 endonuclease VII. J. Mol. Biol. 1995;252:596–610.

Li CL, Hor LI, Chang ZF, Tsai LC, Yang WZ, Yuan HS. DNA binding and cleavage by the periplasmic nuclease Vvn: A novel structure with a known active site. EMBO J. 2003;22:4014-4025.

Palanivelu P. DNA polymerases – An insight into their active sites and mechanism of action. In: Recent Advances in Biological Research, SCIENCEDOMAIN International Book Publishers, UK. 2019;1: 1-39. ISBN: 9788193422441.

DOI: 10.9734/bpi/rabr/v1

Selent U, Rueter T, Koehler E, Liedtke M, Thielking V, Alves J, Oelgeschlaeger T, Wolfes H, Peters F, Pingoud A. A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV. Biochemistry. 1992;31:4808-4815.

Fonfara I, Le Rhun A, Chylinski K, Makarova KS, Lecrivain AL, Bzdrenga J, Koonin EV, Charpentier E. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 2014;42: 2577–2590.

Anders C, Niewoehner O, Duerst A, Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014;513:569–573.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier EA. Programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816–821.