#endonuclease

Processing of rRNA and tRNA

Homing endonuclease recognizes

Homing endonuclease recognizes an extended homing site, typically 14-40 base pairs, corresponding exactly to the intron insertion locus in intron-free allele. Recognition sequence spans cleavage site, ensuring endonuclease cuts only alleles lacking intron, because presence of intron disrupts site. Cleavage generates 4-base 3' overhangs or blunt ends, activating homologous recombination machinery. High specificity prevents widespread genome toxicity yet tolerates degeneracy for spread to related sites. Cellular repair copies intron plus flanking homology, propagating element efficiently. This refined regulation supports accurate ribosomal assembly, quality control and translational fidelity under diverse physiological conditions and growth states.

Ref: NCBI Bookshelf, Molecular Biology of the Cell, Section: Homing endonuclease recognition of DNA homing site

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Transcript analysis

Which of the following statements is true for S1 endonuclease?

S1 nuclease, purified from Aspergillus oryzae, is a single-strand specific endonuclease widely used in transcript analysis. It hydrolyzes phosphodiester bonds in single-stranded DNA and RNA, degrading unpaired regions. Double-stranded DNA with nicks, gaps, or loops also serves as substrate because local single-stranded character is exposed. It does not cleave fully base-paired duplexes. This property underpins S1 nuclease protection assay, where RNA-DNA hybrids are protected while unhybridized single-stranded tails are digested, allowing precise mapping of transcript termini and intron-exon boundaries accurately.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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RDT Basics

DNase I is a type of:

Deoxyribonuclease I is an endonuclease that hydrolyzes internal phosphodiester bonds rather than removing nucleotides from termini. Isolated from bovine pancreas, it is a 31 kilodalton glycoprotein requiring divalent cations, specifically calcium for stability and magnesium for catalytic activity, yielding fragments with 5'-phosphate and 3'-hydroxyl ends. It degrades double-stranded DNA, with slower activity on single-stranded DNA and chromatin. Limiting enzyme concentration generates single-strand nicks useful for nick translation labeling, while higher concentrations produce double-strand breaks. Its endonucleolytic mechanism contrasts with exonucleases acting from ends.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Gene Tar getting

Cas9 endonuclease cuts the DNA and creates:

Cas9 is dual nuclease utilizing HNH domain to cut target strand complementary to guide RNA and RuvC domain to cut non-target strand simultaneously. Both nicks occur three base pairs upstream of PAM on opposite strands, generating predominantly blunt ended double strand breaks without single stranded overhangs. Such blunt breaks are predominantly repaired by non-homologous end joining causing small indels, or by homology directed repair if donor template provided. Blunt nature facilitates efficient ligation mediated insertion and predictable editing outcomes in genome engineering laboratories worldwide.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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