Skip to content
Login Register

#DNA cleavage

6 public questions tagged with this topic.

Transgenic Plant -2

FokI nuclease requires __________ for DNA cleavage.

FokI restriction enzyme from Flavobacterium okeanokoites is Type IIS enzyme recognizing GGATG and cutting 9/13 downstream, separated into N-terminal DNA recognition domain and C-terminal cleavage domain. Nuclease domain isolated for genome editing displays catalytic motif PD...D/EXK coordinating Mg2+ for phosphodiester hydrolysis. Critical feature discovered by crystallography is cleavage domain inactive as monomer because catalytic center requires dimerization to align two active sites across DNA duplex forming DSB. Biochemical assays show monomeric FokI binds but cannot incise even with Mg2+. In ZFN context, two monomers must bind in close proximity with spacer length 5-7 bp optimal allowing C-terminal domains to interact via hydrophobic interface. Dimerization requirement provides double checkpoint enhancing specificity, since single ZFN binding does not lead to breakage unlike monomeric nucleases. Engineering obligate heterodimer FokI variants DD: ELAN and RR:DRSH introducing electrostatic repulsion for homodimers further reduces off-target activity, improving therapeutic window. Same principle exploited in TALEN and dimeric Cas9-FokI systems where spatial proximity essential for catalysis ensuring genome stability.

Ref: Bitinaite et al. PNAS 1998 95:10570 FokI dimer; Miller Nat Biotech 2007 Heterodimer; Waugh 1993.

View Details
Recombination

HO endonuclease cleaves

Mating type switching requires precise double-strand break initiation at active MAT locus. HO endonuclease is highly specific endonuclease expressed transiently in mother cells during late G1. It recognizes long degenerate 24 base-pair site spanning Ya/Z1 region at MAT, cleaving to generate 4 nucleotide 3' overhangs. Following cleavage, resection proceeds and strand invades silent donor cassette. HML and HMR themselves resist HO cleavage because heterochromatin and Sir proteins protect and because cleavage site degraded after donor copying. Thus HO activity is restricted to MAT enabling controlled conversion.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 7: HO Endonuclease Cleavage at MAT Locus

View Details
Sequencing

Hydrazine without salt cleaves which bases?

Chemical sequencing with hydrazine exploits differential reactivity of pyrimidines. Hydrazine opens pyrimidine rings, rendering them susceptible to piperidine-mediated backbone scission. The reaction outcome depends on salt concentration. Hydrazine without added sodium chloride modifies both thymine and cytosine, yielding a combined T plus C cleavage lane. Adding high salt such as NaCl or using prolonged conditions suppresses thymine reaction, leaving cytosine-specific cleavage. Comparison of two lanes allows distinction between T and C. Purines A and G remain unreactive to hydrazine; they require other chemicals.

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.

View Details
Restriction Digestion & Mapping

Which enzyme creates sticky ends?

EcoRI recognizes GAATTC and creates a staggered cut between G and A on both strands, leaving AATT 5' overhangs that are single-stranded and complementary, termed sticky or cohesive ends. DNase I is a nonspecific endonuclease that degrades DNA randomly. Taq and Pfu polymerases are thermostable DNA synthesizers used in PCR, not restriction enzymes, and they add non-templated A or produce blunt products respectively. Therefore only EcoRI among listed creates characteristic cohesive termini useful for directional cloning and efficient ligation via complementary base pairing.

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.

View Details
Apoptosis

DNA laddering during apoptosis occurs due to cleavage at

Linker DNA between nucleosomes, is consistent with established principles of cell signaling, receptor pharmacology and cellular regulation. Experimental measurements of binding parameters, genetic loss-of-function studies and pharmacological interventions all converge on the same interpretation. Related options address neighboring concepts but do not satisfy the precise criterion stated in the question.

Ref: NCERT Biology Class 11–12 Alberts et al Molecular Biology of the Cell Lodish et al, Molecular Cell Biology Cooper & Hausman, The Cell Abbas et al., Cellular and Molecular Immunology (for immunology sections)

View Details