DNA polymerase I (or Pol I) is an enzyme that participates in the process of prokaryotic DNA replication. Discovered by Arthur Kornberg in 1956, it was the first known DNA polymerase (and the first known of any kind of polymerase). It was initially characterized in E. coli and is ubiquitous in prokaryotes. In E. coli and many other bacteria, the gene that encodes Pol I is known as polA. The E. coli Pol I enzyme is composed of 928 amino acids, and is an example of a processive enzyme — it can sequentially catalyze multiple polymerisation steps without releasing the single-stranded template. The physiological function of Pol I is mainly to support repair of damaged DNA, but it also contributes to connecting Okazaki fragments by deleting RNA primers and replacing the ribonucleotides with DNA.

In 1956, Arthur Kornberg and colleagues discovered Pol I by using Escherichia coli (E. coli) extracts to develop a DNA synthesis assay. The scientists added ¹⁴C-labeled thymidine so that a radioactive polymer of DNA, not RNA, could be retrieved. To initiate the purification of DNA polymerase, the researchers added streptomycin sulfate to the E. coli extract. This separated the extract into a nucleic acid-free supernatant (S-fraction) and nucleic acid-containing precipitate (P-fraction). The P-fraction also contained Pol I and heat-stable factors essential for the DNA synthesis reactions. These factors were identified as nucleoside triphosphates, the building blocks of nucleic acids. The S-fraction contained multiple deoxynucleoside kinases. In 1959, the Nobel Prize in Physiology or Medicine was awarded to Arthur Kornberg and Severo Ochoa "for their discovery of the mechanisms involved in the biological synthesis of Ribonucleic acid and Deoxyribonucleic Acid."

Pol I mainly functions in the repair of damaged DNA. Structurally, Pol I is a member of the alpha/beta protein superfamily, which encompasses proteins in which α-helices and β-strands occur in irregular sequences. E. coli DNA Pol I consists of multiple domains with three distinct enzymatic activities. Three domains, often referred to as thumb, finger and palm domain work together to sustain DNA polymerase activity. A fourth domain next to the palm domain contains an exonuclease active site that removes incorrectly incorporated nucleotides in a 3' to 5' direction in a process known as proofreading. A fifth domain contains another exonuclease active site that removes DNA or RNA in a 5' to 3' direction and is essential for RNA primer removal during DNA replication or DNA during DNA repair processes.

E. coli bacteria produces 5 different DNA polymerases: DNA Pol I, DNA Pol II, DNA Pol III, DNA Pol IV, and DNA Pol V.

In DNA replication, the leading DNA strand is continuously extended in the direction of replication fork movement, whereas the DNA lagging strand runs discontinuously in the opposite direction as Okazaki fragments. DNA polymerases also cannot initiate DNA chains so they must be initiated by short RNA or DNA segments known as primers. In order for DNA polymerization to take place, two requirements must be met. First of all, all DNA polymerases must have both a template strand and a primer strand. Unlike RNA, DNA polymerases cannot synthesize DNA from a template strand. Synthesis must be initiated by a short RNA segment, known as RNA primer, synthesized by Primase in the 5' to 3' direction. DNA synthesis then occurs by the addition of a dNTP to the 3' hydroxyl group at the end of the preexisting DNA strand or RNA primer. Secondly, DNA polymerases can only add new nucleotides to the preexisting strand through hydrogen bonding. Since all DNA polymerases have a similar structure, they all share a two-metal ion-catalyzed polymerase mechanism. One of the metal ions activates the primer 3' hydroxyl group, which then attacks the primary 5' phosphate of the dNTP. The second metal ion will stabilize the leaving oxygen's negative charge, and subsequently chelates the two exiting phosphate groups.

The X-ray crystal structures of polymerase domains of DNA polymerases are described in analogy to human right hands. All DNA polymerases contain three domains. The first domain, which is known as the "fingers domain", interacts with the dNTP and the paired template base. The "fingers domain" also interacts with the template to position it correctly at the active site. Known as the "palm domain", the second domain catalyses the reaction of the transfer of the phosphoryl group. Lastly, the third domain, which is known as the "thumb domain", interacts with double stranded DNA. The exonuclease domain contains its own catalytic site and removes mispaired bases. Among the seven different DNA polymerase families, the "palm domain" is conserved in five of these families. The "finger domain" and "thumb domain" are not consistent in each family due to varying secondary structure elements from different sequences.

Pol I possesses four enzymatic activities:

In order to determine whether Pol I was primarily used for DNA replication or in the repair of DNA damage, an experiment was conducted with a deficient Pol I mutant strain of E. coli. The mutant strain that lacked Pol I was isolated and treated with a mutagen. The mutant strain developed bacterial colonies that continued to grow normally and that also lacked Pol I. This confirmed that Pol I was not required for DNA replication. However, the mutant strain also displayed characteristics which involved extreme sensitivity to certain factors that damaged DNA, like UV light. Thus, this reaffirmed that Pol I was more likely to be involved in repairing DNA damage rather than DNA replication.