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#nick translation

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Replication in Prokaryotes

Nick translation is performed by

Nick translation is coupled exonuclease and polymerase reaction moving nick position along duplex without net synthesis. Pol I binds nick bearing free 3' OH, degrades downstream strand via 5' to 3' exonuclease while extending 3' end with polymerase activity incorporating new dNTPs, effectively translating nick along DNA. Property historically exploited to label DNA probes with radioactive dNTPs replacing strand segment uniformly. Pol III cannot degrade downstream duplex, ligase only seals nicks without synthesis. In vivo role of Pol I during Okazaki fragment processing directly mirrors laboratory nick translation mechanism.

Ref: Berg et al., Biochemistry, 9th ed., Chapter 27: Nick translation mechanism by DNA polymerase I

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Nucleic acid probe and labelling

Which labeling technique uses nicking of DNA followed by polymerase activity?

Nick translation creates labeled DNA by mimicking cellular DNA repair synthesis. DNase I at low concentration introduces single-strand breaks by cleaving phosphodiester bonds, leaving 3'-hydroxyl and 5'-phosphate ends. Escherichia coli DNA polymerase I then binds at nicks; its 5' to 3' exonuclease activity removes nucleotides ahead while its polymerase activity incorporates new nucleotides, including labeled deoxynucleotides, behind. The nick thus translates along the duplex. This coordinated excision and synthesis yields uniformly labeled double-stranded probes with high specific activity for in situ hybridization and membrane blotting.

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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