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This page is general reference information for researchers and laboratory staff, not medical advice.
Short answer: what does DNA polymerase I do?
DNA polymerase I (Pol I, also written DNA polymerase 1 or DNA pol I) is the E. coli enzyme encoded by polA. It adds nucleotides to a primed DNA template in the 5’→3′ direction and also carries two exonuclease activities (3’→5′ and 5’→3′), which lets it fill gaps and nicks during DNA repair, recombination and replication. Its proteolytic large fragment, the Klenow fragment, keeps the polymerase and 3’→5′ exonuclease activities but loses the 5’→3′ exonuclease, and that difference decides which one a bench application uses.
Definition and scope
UniProt entry P00582 describes E. coli DNA polymerase I as a single-chain monomer with multiple functions, classified as EC 2.7.7.7 and belonging to the DNA polymerase type-A family. In addition to polymerase activity it shows 3’→5′ and 5’→3′ exonuclease activity, can use nicked circular duplex DNA as a template and can unwind the parental strand from its template. The Worthington Enzyme Manual credits the enzyme’s discovery to Arthur Kornberg and colleagues in 1956 and gives a molecular weight of about 109 kDa.
Five distinct DNA polymerases have been isolated from E. coli, designated I, II, III, IV and V (Worthington Enzyme Manual). Pol I is described there as the predominant polymerizing enzyme in E. coli, with the job of filling DNA gaps that arise during replication, repair and recombination. The names “DNA polymerase 1”, “DNA pol 1”, “DNA polymerase I” and “Pol I” all refer to this same enzyme.
How it works: the three activities
5’→3′ polymerase activity
Per the Worthington manual, synthesis requires a primer with a free 3′-OH annealed to a template; nucleotides are added in the 5’→3′ direction and pyrophosphate is released. The enzyme is active on DNA that contains single-stranded gaps and on DNA that contains nicks.
3’→5′ exonuclease activity
UniProt lists a 3’→5′ exonuclease activity for Pol I.
5’→3′ exonuclease activity
The Worthington manual states that this activity degrades DNA in the 5’→3′ direction, giving 5′-mononucleotides, and can excise mismatched regions. Combined with polymerization at the same nick, it is the basis of nick translation.
Klenow fragment: Pol I with one activity removed
The Worthington manual defines the Klenow fragment as a proteolytic product of E. coli DNA polymerase I that retains polymerization and 3’→5′ exonuclease activity but has lost 5’→3′ exonuclease activity. It runs at roughly 70 kDa by gel filtration, against about 109 kDa for the full enzyme. The manual dates the proteolytic work that produced the large fragment to a 1970 reference involving Klenow and colleagues.
Activity map and bench uses: Pol I, Klenow fragment and Taq
Which polymerase fits a task depends on which activities the task needs and which it cannot tolerate. The table combines the UniProt and Worthington statements above with UniProt entry P19821 for Thermus aquaticus (Taq) DNA polymerase I.
| Enzyme | 5’→3′ polymerase | 3’→5′ exonuclease | 5’→3′ exonuclease | Uses named in the sources |
|---|---|---|---|---|
| E. coli DNA polymerase I | Yes | Yes | Yes | Nick translation labelling of DNA (Worthington) |
| Klenow fragment | Yes | Yes | No (lost) | Sanger DNA sequencing (1977); filling in 5′ overhangs or removing 3′ overhangs to make blunt ends; second-strand synthesis in mutagenesis (Worthington) |
| Taq DNA polymerase I (T. aquaticus) | Yes | Unlikely: UniProt notes no 3’→5′ exonuclease domain | Yes | UniProt links the missing proofreading to a relatively high error rate |
Reading the table as a decision aid: an application that needs the 5’→3′ exonuclease at a nick (nick translation) calls for full-length Pol I, while an application that must not degrade the 5′ strand downstream of the primer (blunt-ending, second-strand synthesis) calls for the Klenow fragment, which has lost that activity. For the thermostable enzyme used in amplification, see the related thermal cycler guide and the PCR protocol basics guide.
DNA polymerase I vs II vs III in E. coli
The three best-known E. coli polymerases differ in role, not in the basic chemistry (all three are listed under EC 2.7.7.7 in UniProt).
| Enzyme | Role stated by the source | Source |
|---|---|---|
| DNA polymerase I | Fills DNA gaps arising during replication, repair and recombination; predominant polymerizing enzyme in E. coli | Worthington; UniProt P00582 |
| DNA polymerase II | Thought to be involved in DNA repair and/or mutagenesis; processivity enhanced by the beta sliding clamp (dnaN) and clamp loader | UniProt P21189 |
| DNA polymerase III | A complex, multichain enzyme responsible for most of the replicative synthesis in bacteria; alpha chain is the catalytic subunit; also has 3’→5′ exonuclease activity | UniProt P10443 |
UniProt adds that Pol III is tethered to replicating DNA by the beta sliding clamp, which gives the catalytic subunit extremely high processivity: it can copy a 5.4 kb genome in 11 seconds, at least 500 nucleotides per second at 30 °C. Pol I, by contrast, is described as a single-chain monomer that fills gaps.
Where it matters for labs
For a laboratory, the practical point is enzyme selection. The sources above tie each application to a specific set of activities, so the table can be read from the application backwards: decide which activities the task needs, then match the enzyme. See also the guides on DNA sequencing and Sanger sequencing (historically a Klenow application per the Worthington manual), restriction enzyme digests that leave overhangs, and site-directed mutagenesis.
Common misunderstandings
- “Pol I is the main replicative polymerase.” UniProt assigns most replicative synthesis in bacteria to DNA polymerase III; Pol I fills gaps.
- “Klenow fragment is a different enzyme.” It is a proteolytic product of Pol I that has lost its 5’→3′ exonuclease activity.
- “All polymerases proofread.” UniProt notes Taq polymerase is unlikely to have 3’→5′ exonuclease activity.
- “Polymerase 1 and RNA polymerase I are the same.” They are not; see the FAQ.
FAQ
What does DNA polymerase 1 do?
It synthesizes DNA 5’→3′ from a primer, and its exonuclease activities let it remove nucleotides ahead of or behind the growing end. In E. coli this fills gaps arising in replication, repair and recombination (Worthington Enzyme Manual).
What is the difference between DNA polymerase 1 and 3?
Pol III is the multichain replicative enzyme responsible for most replicative synthesis in bacteria, made processive by the beta sliding clamp. Pol I is a single-chain enzyme that fills gaps.
What is the difference between DNA polymerase 1 and 2?
UniProt describes Pol II as thought to be involved in DNA repair and/or mutagenesis, with processivity enhanced by the beta clamp and clamp loader. Pol I is the gap-filling enzyme with the 5’→3′ exonuclease activity.
What are the five DNA polymerases?
In E. coli, five distinct DNA polymerases are designated I, II, III, IV and V (Worthington Enzyme Manual).
What is the main function of DNA polymerase 3?
Most of the replicative synthesis in bacteria; the alpha chain is the catalytic subunit (UniProt P10443).
What are the core subunits of DNA polymerase III?
UniProt identifies the alpha chain (1,160 amino acids in E. coli) as the catalytic subunit of a multichain complex, tethered by the beta clamp (dnaN).
What are polymerases?
Enzymes that catalyze template-directed synthesis of nucleic acid strands. DNA polymerases make DNA (EC 2.7.7.7: DNA(n) + a deoxyribonucleoside 5′-triphosphate gives DNA(n+1) + diphosphate).
What is the function of RNA polymerase 1?
RNA polymerase I is a different enzyme. Per UniProt (human RPA1, O95602), it synthesizes ribosomal RNA precursors, transcribing 47S pre-rRNA that gives rise to 5.8S, 18S and 28S rRNAs.
Sources
- UniProt P00582, E. coli DNA polymerase I, rest.uniprot.org/uniprotkb/P00582, read 2026-11-07.
- UniProt P21189 (DNA polymerase II), P10443 (DNA polymerase III subunit alpha), P19821 (T. aquaticus DNA polymerase I) and O95602 (RNA polymerase I subunit RPA1), read 2026-11-07.
- Worthington Biochemical, Enzyme Manual: DNA Polymerase I, worthington-biochem.com/products/dna-polymerase-i/manual, read 2026-11-07.
