Nick translation is a molecular biology technique and a natural enzymatic process involving DNA polymerase I. It plays a crucial role in DNA repair and labeling by replacing nucleotides at single-stranded breaks or nicks in double-stranded DNA. This process exploits the unique activities of DNA polymerase I, which can simultaneously remove nucleotides ahead of a nick and add new nucleotides behind it, effectively “translating” the nick along the DNA strand.

What Is a Nick in DNA?

A nick is a discontinuity in one strand of double-stranded DNA where the phosphodiester bond between adjacent nucleotides is missing. This break leaves a free 3′-hydroxyl (OH) group and a 5′-phosphate group on the DNA backbone but does not disrupt the complementary strand.

Nicks can arise naturally during DNA metabolism or be introduced experimentally by nucleases such as DNase I.

Mechanism of Nick Translation

1. Nick Formation:
   DNA is treated with an enzyme like DNase I to introduce single-stranded nicks at random or specific locations.

2. DNA Polymerase I Action:
   DNA polymerase I binds at the nick and performs two simultaneous activities:

   • 5′→3′ exonuclease activity: Removes nucleotides ahead of the nick on the 5′ side.

   • 5′→3′ polymerase activity: Adds new nucleotides to the free 3′-OH end behind the nick.

3. Translation of the Nick:
   As nucleotides are removed and replaced, the nick effectively moves or “translates” along the DNA strand in the 3′ direction.

4. Result:
   The original nucleotides at the nick are replaced by new nucleotides, which can be labeled (radioactively or fluorescently) if modified nucleotides are supplied.

5. Nick Sealing:
   DNA ligase can later seal the remaining nick to restore the continuous phosphodiester backbone.

Applications of Nick Translation

• DNA Labeling:
  Incorporation of labeled nucleotides (radioactive or fluorescent) during nick translation allows the preparation of probes for techniques such as Southern blotting, fluorescence in situ hybridization (FISH), and other hybridization assays.

• DNA Repair:
  Nick translation is part of the natural DNA repair process, where damaged or mismatched nucleotides are excised and replaced.

• Molecular Biology Research:
  Used to generate labeled DNA fragments for tracking, detection, or studying DNA-protein interactions.

Why Nick Translation Is Important

• It enables precise replacement of nucleotides at specific DNA sites.

• Facilitates the creation of uniformly labeled DNA probes with high specific activity.

• Supports the study of DNA structure, function, and interactions in various biological contexts.

Clarifying the Given Options

• (1) Translation by cytosolic ribosome: Incorrect; translation refers to protein synthesis, unrelated to nick translation.

• (2) Translation of protein from stalled sites: Incorrect; nick translation is a DNA process, not protein synthesis.

• (3) Replication by DNA polymerase I after removing RNA primers: Partially related but not the definition of nick translation. This describes DNA polymerase I function in Okazaki fragment processing, not nick translation per se.

• (4) Replication of DNA by DNA polymerase I from nicks produced by DNase treatment: Correct; this describes the nick translation process where DNA polymerase I extends from nicks created by DNase, removing and replacing nucleotides.

Final Answer

(4) Replication of DNA by DNA polymerase I from nicks produced by DNase treatment

Keywords

nick translation, DNA polymerase I, 5′→3′ exonuclease, DNA labeling, DNA repair, Okazaki fragments, DNase I, DNA probes, fluorescence in situ hybridization, molecular biology techniques

Summary:
Nick translation is a process where DNA polymerase I removes nucleotides at single-stranded nicks in DNA and replaces them with new nucleotides, effectively moving the nick along the strand. This mechanism is exploited in molecular biology for labeling DNA and is essential in DNA repair pathways.