#DNA polymerase I

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 laborat

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

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Replication in Prokaryotes

Klenow fragment lacks which activity?

Limited proteolysis of Pol I by subtilisin yields large C-terminal Klenow fragment comprising polymerase and 3' to 5' exonuclease domains, removing N-terminal 5' to 3' exonuclease domain. Klenow retains templated synthesis and proofreading, widely used historically for DNA labeling, second strand cDNA synthesis, and Sanger sequencing before recombinant enzymes available. Loss of 5' to 3' activity prevents unwanted degradation of template during synthesis. Modern Klenow exo-minus version also lacks proofreading for higher efficiency incorporation of modified nucleotides and dideoxy terminators.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Tool Box: Klenow fragment preparation and activities, proteolysis of Pol I

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Replication in Prokaryotes

5′→3′ exonuclease activity is present in

DNA polymerase I is unique among E. coli polymerases for possessing two distinct exonuclease activities within one polypeptide. Its 3' to 5' proofreading domain removes misincorporated nucleotides from growing strand, while N-terminal domain carries 5' to 3' exonuclease that cleaves RNA primers and downstream DNA during nick translation. Pol II possesses only 3' to 5' activity, Pol III epsilon provides proofreading but no 5' to 3' function. Dual nuclease capability qualifies Pol I for Okazaki fragment maturation, gap filling, and base excision repair patch removal.

Ref: NCBI Bookshelf, Molecular Biology of Escherichia coli, DNA Pol I domain structure and exonuclease activities

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Replication in Prokaryotes

DNA polymerase I was discovered by

In 1956 Arthur Kornberg at Washington University isolated enzymatic activity from E. coli extracts that incorporated labeled dNTPs into DNA requiring primer, template, and Mg2+. This activity now called DNA polymerase I provided first biochemical proof of enzymatic DNA synthesis in vitro. Kornberg received 1959 Nobel Prize in Physiology for this discovery. His son Thomas Kornberg later discovered Pol III replicase. Original assay using incorporation of 14C thymine and defined activated DNA template-primer paved way for subsequent discovery of replication fork proteins mechanisms and regulation

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Historical: Discovery of DNA polymerase I by Arthur Kornberg, 1956

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