DNA polymerase

DNA polymerase(s) refers to a group of polymerases that catalyze the synthesis of polydeoxyribonucleotides from mono-deoxyribonucleoside triphosphates (dNTPs) and perform the basic functions of DNA replication, repair and, in some cases, cell differentiation in vivo. DNA polymerases play a central role in DNA replication, i.e. the production of identical copies of DNA.

Five DNA polymerases are found in humans (and all other mammals): α, β, γ, δ and ε. It can be assumed that the polymerases δ and ε are crucial for replication, as they are characterized by high processivity and proof reading function. In contrast, the polymerases α and β show only low processivity and no proofreading function.

There are also RNA-dependent DNA polymerases that use RNA as a template and attach dNTPs. These are known as reverse transcriptases. Telomerase also belongs to this group. Only the terminal deoxyribonucleotidyltransferase is known as an independent DNA polymerase.

In addition to the polydeoxyribonucleotides (dNTPs), DNA polymerases require an initiating oligonucleotide (or polynucleotide), a so-called primer, which carries a hydroxyl group at the 3′ end that can be used as a starting point for chain growth. DNA polymerases cannot initiate the synthesis of mononucleotides de novo. A primer can be a short or long piece of DNA or RNA that carries a free 3′-OH group. Primers provide the DNA polymerase with a double-stranded structure by annealing to a complementary region of the DNA or RNA strand, the so-called template. The DNA polymerase moves along the DNA (or RNA) template and extends the primer in the 5′ → 3′ direction according to the Watson-Crick base pairing rule, i.e. A pairs with T (or U) and C with G. The polarity of the newly synthesized chain is opposite (or antiparallel) to that of the template. The insertion of a non-complementary nucleotide is considered an "error". The error frequency is an important characteristic of a polymerase.

In addition to the main 5′ → 3′ polymerase activity, a DNA polymerase may have several other activities, e.g. 5′ nuclease, 3′ → 5′ exonuclease and/or RNase H activities, which are required for proper function in vivo.

Template-dependent DNA polymerases generally possess multiple domains that have distinct activities but function in vivo in a coordinated manner to ensure high polymerization rate, maximal replication fidelity, and regulation of polymerase activity. The most common combination of multifunctions is polymerase and 3′ → 5′-exonuclease activity, as found in the DNA polymerases of E. coli, T4 and T7.

Another common combination is the polymerase and RNase H activities found in reverse transcriptases. Although they are physically and functionally closely linked, each function is located in a separate domain. For example, the 3′ → 5′ exonuclease and/or RNase H activities can be selectively inhibited or inactivated without affecting the polymerase activity.

In higher eukaryotes, the start of cellular DNA replication takes place at several specific sites on the DNA, which are referred to as replication origins (Ori). The site of dsDNA where replication occurs is called the "replication fork". Due to the polarity of the DNA strands, bidirectional replication leads to two different products, the "leading" and the "lagging" strand, depending on the direction of movement of the replication fork. The leading strand is synthesized as a single continuous chain, while the lagging strand is first synthesized as small oligonucleotides, the so-called Okazaki fragments, which are then ligated into a continuous chain. Small RNAs play an important role as natural primers in the synthesis of both the leading strand and, in particular, the lagging strand.

Functional concepts of polymerase: processivity and fidelity.

The functional roles of DNA polymerases in DNA replication, synthesis and repair are based on a number of important concepts that define the enzymatic properties of polymerases. Two of these concepts that are relevant to the polymerases that catalyze templated DNA synthesis are processivity and fidelity . Processivity and fidelity of a DNA polymerase are not only enhanced by the coordinated actions of polymerase and polymerase-associated activities, but are also modulated by the interactions with other proteins, called accessory proteins, which form a replication complex with the DNA polymerase.

Processivity: A DNA polymerase involved in DNA synthesis can be continuously bound (or not) and move stepwise along the template. In fact, after the addition of a certain number of nucleotides, DNA polymerases drop off the template and reattach to re-initiate polymerization. The rate of DNA chain elongation catalyzed by DNA polymerases is also not uniform. Processivity is the average number of nucleotides added by a polymerase molecule each time it binds a template. Processivity is an intrinsic property of the polymerase. A polymerase is said to be processive if it copies a long template while maintaining uninterrupted contact with it. The number of nucleotides polymerized per binding-dissociation event indicates whether a polymerase has "low" or "high" processivity. Processivity is also influenced by factors that affect the secondary structure of the DNA and the conformation of the enzyme. Processivity is therefore a characteristic that is kinetically related to the properties of the DNA break or termination sites.

Fidelity of DNA polymerase: Another important parameter that characterizes a DNA polymerase is fidelity, i.e. the accuracy of nucleotide incorporation or the frequency of misincorporation. The fidelity of the polymerase can be correlated with the mutation rate typically observed in retroviruses such as HIV. The fidelity of a DNA polymerase is ensured by the coordinated action of polymerase activity and 3′ → 5′-exonuclease activity. The 3′ → 5′-exonuclease activity, also known as proofreading or editing exonuclease, can either be an integral part of the polymerase molecule as in E. coli DNA Pol I or it can be associated with the polymerase as a multi-subunit complex as in E. coli DNA Pol III. Although the exact mechanism remains to be elucidated, it is believed that a polymerase makes the distinction between correct and incorrect nucleotide binding and chemical sealing primarily on the basis of local DNA geometry and, to a lesser extent, the energetics of competing reactions. Therefore, the fidelity of a polymerase is largely due to a kinetic blockade that inhibits the elongation of termini after nucleotide misinsertion.

In biotechnology, DNA synthesis can also be carried out in vitro under suitable technical conditions with a slightly different level of complexity. This process is known as the polymerase chain reaction (PCR).

1. Chatterjee N et al. (2020) A stapled POL kappa peptide targets REV1 to inhibit mutagenic translesion synthesis. Environ Mol Mutagen 61:830-836
2. Ikeh KE et al. (2021) REV1 Inhibition Enhances Radioresistance and Autophagy. Cancers (Basel) 13:5290.
3. Sui X et al. (2013) Autophagy and chemotherapy resistance: A promising therapeutic target for cancer treatment. Cell Death Dis 4:e838.
4. Yamanaka K et al. (2017) Inhibition of mutagenic translesion synthesis: A possible strategy for improving chemotherapy? PLoS Genet 13:e1006842.
5. Li Y et al. (2000) Identification and cloning of two histone fold motif-containing subunits of HeLa DNA polymerase epsilon. J Biol Chem 275:23247-2352.
6. Ogi T et al. (2010) Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells. Mol Cell 37:714-727.
7. Wimmer K et al. (2017) A novel germline POLE mutation causes an early onset cancer prone syndrome mimicking constitutional mismatch repair deficiency. Fam Cancer16: 67-71.