Abstract
DNA polymerases catalyze nucleotidyl transfer, the central reaction in synthesis of DNA polynucleotide chains. They function not only in DNA replication, but also in diverse aspects of DNA repair and recombination. Some DNA polymerases can perform translesion DNA synthesis, facilitating damage tolerance and leading to mutagenesis. In addition to these functions, many DNA polymerases conduct biochemically distinct reactions. This review presents examples of DNA polymerases that carry out nuclease (3ʹ—5′ exonuclease, 5′ nuclease, or end-trimming nuclease) or lyase (5′ dRP lyase) extracurricular activities. The discussion underscores how DNA polymerases have a remarkable ability to manipulate DNA strands, sometimes involving relatively large intramolecular movement.
Introduction
DNA polymerases have been intensively studied for decades because of their fundamental importance in DNA replication. Organisms throughout nature possess an array of polymerases encoded by their genomes, specialized for functions in DNA repair, recombination, and DNA damage tolerance. The canonical DNA polymerase reaction is the addition of a nucleotide, usually a deoxynucleoside triphosphate, to the 3′ end of a growing DNA chain, liberating pyrophosphate (Figure 1). The reverse reaction, pyrophosphorolysis, catalyzed by some DNA polymerases, is driven backwards by an excess of pyrophosphate. The purpose of this review is to highlight the existence of additional activities associated with DNA polymerases beyond canonical nucleotidyl transfer (Figure 2). We briefly summarize each activity, emphasizing recent results and unsolved issues.
FIGURE 1
FIGURE 2
3′–5′ Exonuclease Activity
All DNA polymerases share a common polymerase fold, which has been compared to a human right hand, composed of three subdomains: fingers, palm, and thumb (). In addition, the DNA polymerase toolbox can include a 3′–5′ exonuclease domain whose main role is to proofread new DNA synthesis to remove nucleotides that have been incorrectly incorporated. This reduces the error rate of DNA polymerases by one or more orders of magnitude during DNA replication (). In certain contexts, this 3′–5′ exonuclease activity is essential. Loss of editing activity results in increased incidence of cancer and pronounced genome instability (; ).
Multiple families of DNA polymerases harbor a 3′–5′ exonuclease activity: B-, C-, and D-family replicative polymerases (δ and ε in eukaryotes, Pol III in E. coli, PolD in euryarchaeota) and A-family repair polymerases, such as E. coli Pol I (). Many A-family and some B-family polymerases have a conserved 3′–5′ exonuclease domain located in the N-terminal region of the larger polymerase domain (). In other B-family polymerases, the location of the exonuclease domain is found in a structurally distant region. The exonuclease active site in E. coli DNA polymerase I, an A-family polymerase, is ∼30 Å away from the polymerase active site (Figure 3A), whereas the distance between the two active sites is closer to ∼40 Å in B-family polymerases (Figure 3B) (; ). Most B-family DNA polymerases have an extended β-hairpin loop that facilitates primer movement by holding the template strand in place as the primer switches into the exonuclease active site, a feature not found in A-family polymerases with 3′–5′ exonuclease activity ().
FIGURE 3
Although homology among DNA polymerases varies significantly, the 3′–5′ exonuclease active site itself is very conserved within A-family polymerases. There are three highly conserved amino acid regions containing critical residues that coordinate two divalent metal ions, ssDNA and deoxyribonucleoside monophosphates (dNMPs) (; ). A key catalytic step required for 3′–5′ exonuclease activity is the chelation of two metal ions by active-site aspartates and glutamates (carboxylates) (). The Exo I, Exo II, and Exo III regions are essential for the exonuclease activity, as they contain the carboxylates that directly bind the metal ions required for catalysis (). Recent studies have identified other residues that help facilitate catalysis in the exonuclease active site. In bacteriophage ϕ29 DNA polymerase, tyrosine, threonine, and glutamine residues help coordinate the binding of DNA to the exonuclease active site. Specifically, Tyr 101 and Thr 189 assist in melting dsDNA to form ssDNA that can bind the active site (Rodriguez et al., 2019). Although Thermus aquaticus DNA polymerase (Taq) comprises an exonuclease domain, the enzyme lacks the three aspartates and one glutamate essential for catalysis and therefore has no proofreading activity (); nevertheless exonuclease activity can be conferred to some extent by introducing the four carboxylates into the Taq sequence ().
To ensure replication fidelity the exonuclease activity operates in a delicate balance with the polymerase activity, with the exonuclease favored when a mismatch is inserted (). The enzyme contributes to this regulation by monitoring the newly incorporated bases. The polymerase senses a mismatch by using strategically positioned protein residues that detect the minor groove N3 and exocyclic O2 positions of incorrect base pairs after incorporation, such as Arg 429 and Gln 615 in T7 DNA polymerase, and Tyr 567 and Lys 706 in RB69 gp43, respectively (; ; ). In Family B DNA polymerase ϵ, conserved Lys 967 and Arg 988 were found essential in regulating the switching of the DNA primer between the polymerase active site and exonuclease active site. Both residues lie between the palm and thumb domains and directly interact with the minor groove (). When a mismatch is present, the melting temperature of the bases at the 3′ terminus is lower favoring the formation of ssDNA ends. In fact, the binding of at least 3-4 bp ssDNA in the exonuclease active site is required to favor the cleavage reaction over the polymerase reaction, (; ). The functional separation of the two active sites also helps in the regulation of the exonuclease activity, to ensure it is used sparingly and only when mismatches are introduced. To achieve proper binding in the exonuclease active site, the enzyme must translocate backwards, leading to sufficient DNA unwinding to allow the primer terminus to leave the polymerase active site and bind the exonuclease active site (). More recent FRET experiments mapped the trajectories recorded by a DNA primer translocating from the polymerase active site to the exonuclease active site of the Klenow fragment of DNA pol I. These data revealed that the enzyme can shuttle the primer between the two sites without dissociating from DNA (intramolecular transfer) ().
In some B-family polymerases, the two domains may be more structurally distant, complicating this active site switching step. To overcome this obstacle, some polymerases can bend the DNA in the active site to bring the exonuclease domain closer to the polymerase domain, facilitating the cleavage reaction. In the bacteriophage ϕ29 DNA pol, the primer terminus moves in one dimension from the polymerization site to the exonuclease site by a 40° rotation in the helix of the DNA (; ). In addition to the conserved catalytic aspartates in the exonuclease domain discussed above, a non-catalytic conserved aspartate with a regulatory role has recently been identified (). In ϕ29 DNA pol, this residue, Asp 121, is located between the Exo II and Exo III motifs. This Asp facilitates the binding of ssDNA substrates at the 3′–5′ exonuclease active site, but, interestingly, it does not come into contact with the metal ions or the ssDNA. Asp 121 facilitates the melting of dsDNA to form the ssDNA primer required to catalyze the cleavage reaction. Similar invariant aspartates have also been identified in other enzymes, such as Asp 150 in E. coli RNase T (). In bacteriophage T7 DNA pol, mutating the predicted invariant Asp90 in the active site of the exonuclease domain completely ablated the exonuclease activity of the enzyme (). The proximity of Asp 90 to multiple residues required for the exonuclease reaction suggested to the authors that this residue functions in a “Romanesque vault” structure, facilitating bending of the DNA to bring the primer into the correct orientation in the active site for cleavage ().
Even further redundancies and regulators have been found that facilitate the switch between polymerase activity and exonuclease activity, which is essential for replication fidelity. The switch between polymerase active site and exonuclease active site is also controlled by the concentration of dNTP present in the active site (). Decreased occupancy of the polymerase active site by incoming dNTP shifts the balance towards exonuclease catalysis. More recently, it was shown that phosphorylation of yeast Pol ε during replication fork stalling can also shift the equilibrium. Checkpoint kinases mediate the phosphorylation of Ser 430 in S. cerevisiae pol2, preventing primer DNA switching to exonuclease sites. This post-translational modification regulates the DNA switching between the polymerase active site and the exonuclease site, limiting fork resection and subsequent collapse ().
Beyond proofreading, polymerase 3′–5′ exonuclease activity is important in promoting virus genetic recombination, as seen in Poxviruses. Vaccinia DNA Polymerase mutants lacking 3′–5′ exonucleolytic activity showed a dramatic reduction in recombination (). The authors propose a model in which the polymerase may use 3′–5′ exonuclease activity to rescue a double-strand break (DSB) by exposing sufficient homology in ssDNA to allow annealing at the break, repair by DNA polymerase and ligase, and subsequent resumption of DNA replication ().
5′-Nuclease Activity
Some DNA polymerases, such as Taq, also possess a 5′- nuclease activity located in a separate domain (Figure 4) (; ). The 5′-nuclease activity was originally referred to as a 5′–3′ exonuclease activity, but it has since been established as a structure specific-cleavage of a 5′-ssDNA end joined to duplex DNA, a structure that is formed during lagging strand DNA synthesis (). Conserved residues in the 5’ nuclease domain () share homology with the flap endonuclease (FEN1/XPG) family of proteins (; ; ; ). In E. coli Pol I, the 5′-nuclease domain is tethered to the polymerase domain by an unstructured 16 amino acid peptide that is susceptible to proteolytic cleavage, allowing isolation of a Pol I “Klenow fragment” that lacks the 5′-nuclease domain (). As such, the Pol I 5′-nuclease primarily operates during lagging strand synthesis to remove 5′ flaps formed when Pol I encounters Okazaki fragments during synthesis. The 5′-nuclease activity recognizes the 5′ flap and cuts between the first two bases, creating a nick on the DNA that is subsequently filled by DNA ligase (; ).
FIGURE 4
DNA travels between the polymerase and 5′- nuclease domains through several conformational transitions (). The 5′-nuclease domain is flexible and adopts different positions within the Pol I-DNA complex to remove 5′ flaps (). The polymerase and 5′ nuclease active sites compete for access to DNA ends, with dramatic shifts between the two active sites occurring during ongoing lagging strand synthesis. The 5’ nuclease activity also requires 2 divalent metal ions for cleavage. The selection of activity is likely intricately regulated by molecular “gates” in the active site, although details of this regulation are unknown (). However, the intricate gating mechanisms in FEN family proteins that help discern between different substrates can be used to glean insights into how 5′- nuclease is regulated in Pol I (; ).
5′-dRP Lyase Activity
Steps in base excision repair (BER) occur in a coordinated manner and enzymes are sequentially displaced to facilitate the next step in the pathway. During BER, a damaged base (for example, one modified by reactive oxygen species) is removed by a lesion-specific DNA glycosylase. This process leaves an apurinic/apyrimidinic (AP) site or abasic site that is recognized and cleaved by an AP endonuclease. This cleavage leaves a 5′-terminal deoxyribophosphate (dRP) residue that must be removed in concert with replacement of the base. In mammalian cells, Pol β is the main DNA polymerase involved in BER, and it is able to carry out both of these enzymatic steps. Pol β removes 5′- dRP and fills the gap (; ; ).
The 5′-dRP lyase activity is contained within an 8 kDa N-terminal domain of Pol β (; ) (Figure 5A). A metal ion is required for release of the 5′dRP from double-stranded DNA allowing the gap in the DNA to be filled and subsequently ligated ().
FIGURE 5
A nick adjacent to a 5′-dRP residue is a small target, and Pol β must bind non-specifically along the DNA as it searches for the right substrate to cleave. PARP1 has a high affinity for this substrate and may help recruit pol β (; ). Recently, it was found that three lysines in the lyase active site of Pol β facilitate the search for the 5′- flap substrate on the dsDNA and destabilize non-specific DNA binding. Mutating these three lysine residues to alanines in the lyase active site of Pol β (Lys 35, Lys 68, and Lys 72), increased the binding affinity of Pol β for nonspecific DNA, indicating a role of the 5′dRP lyase in the specificity of lesion recognition (). A tumor-associated variant in the N-terminal 8 kDa domain of Pol β retains polymerase activity but is dRP lyase deficient (). DNA methylating agents such as methyl methane sulfonate (MMS) create base lesions that are removed by BER. Fibroblasts of Pol β null mice are hypersensitive to MMS. This sensitivity can be rescued by complementation with the 5′dRP lyase activity of Pol β, without requiring the DNA polymerase activity (). In this case, another DNA polymerase such as pol δ accomplishes gap filling (; ). Furthermore, Pol β 5′dRP lyase activity is suggested to be important in preventing trinucleotide repeat instability. Pol β defective in 5′-dRP lyase activity can be tightly bound to a repair site, forcing slippage that can result in deletions of trinucleotides ().
Other polymerases also carry out 5′-dRP lyase activity. Y-Family pol iota (Pol ι) possesses 5′-dRP lyase activity and can alleviate BER deficiency in vitro (; ). Loss of Pol ι sensitizes the cells to treatment with oxidative damaging agents (). Pol ι accumulates in sites of oxidative DNA damage and associates with BER modulator XRCC1 to facilitate repair. The recruitment of Pol ι to sites of DNA damage in human cells requires the 5-dRP lyase domain of the enzyme. It is not clear whether the dRPase of Pol ι is functionally relevant to its operation in a pathway to counteract DNA replication stress (), involving p53-dependent reactivation of DNA replication forks (; ).
The A-Family polymerase Pol θ also possesses 5′-dRP lyase activity within its polymerase domain. In vitro, Pol θ can participate in BER reactions (). Lys 2383, a residue critical for AP lyase, is also important for the DNA polymerase activity of Pol θ (Figure 5B) (). The 5′-dRP lyase activity of Pol θ can also operate in structurally clustered lesions (). In these clustered regions, Pol β is 15–20 fold slower at excising the 5′-dRP lyase compared to Pol θ, which suggests that the two polymerases may have evolved to take care of a larger variety of substrates, with Pol β being favored for excising lesions with opposite polarity than those excised by Pol θ (). BER is also important for repair of oxidative lesions in mitochondrial DNA. Mitochondrial DNA polymerase y (Pol γ) also possesses 5′-dRP lyase activity, although with a much slower release of the dRP group from the enzyme compared to Pol β. In fact, the 5′-dRP lyase activity in A-family polymerases such as Pol γ, Pol θ, and Pol I is significantly slower than in Pol β, which suggests that this function may not be sufficient to complete repair of abundant AP sites ().
3′-End-Trimming and Single-Strand Extension
A DNA end-trimming activity, distinct from the nuclease activities discussed above, was identified in human Pol θ (). The rapid end-trimming activity acts on single-stranded DNA when placed in a transient and appropriate self-pairing conformation within pol θ (Figure 6). This end-trimming activity is different from 3′ to 5′ exonuclease activity. Although Pol θ contains a proofreading domain, it lacks two of the conserved amino acids necessary for 3′–5′ exonuclease activity. Further, a short loop in the exonuclease-like domain of pol θ would impede access of DNA to the exonuclease active site ().
FIGURE 6
This end-trimming activity shares many of the dependencies and catalytic residues that are required for the DNA polymerase reaction. For example, end-trimming is dependent on the presence of divalent cation (Mg2+ or Mn2+), on catalytic residues including invariant aspartate Asp 2330 that coordinates Mg2+ in the active site, and on the addition of dNTPs (). Catalysis of both extension and nuclease activity from the same active site is plausible in principle because the two-metal ion active sites of nucleotidyl transfer enzymes share close similarities with the active sites of many nucleases ().
Experimentally, dNTP-dependent end-trimming activity is observed with some, but not all single-stranded oligonucleotides. For those substrates that are end-trimmed, a unifying feature is that the 3′ terminus is capable of potential self-pairing with a short sequence within the oligonucleotide, suggesting that a hairpin-type configuration is transiently formed within the active site of pol θ (Figure 6B). On the most-studied substrate, 2 nt are cleaved from the 3’ terminus in an endonucleolytic reaction; on some substrates 1 nt is removed; on others, possibly more than 2 nt may be end-trimmed. The end-trimming configuration appears to be unimolecular, because all substrate is quickly consumed. Further, the proposed end-trimming configuration explains another activity of pol θ, the ability to extend single-stranded DNA substrate.
Pol θ DNA polymerase can extend some single-stranded DNA molecules, an activity that is not shared by other known A-family polymerases (). In the presence of Mg2+, the products observed are both primed and largely templated by the same oligonucleotide (Figure 6C) (; ). It was suggested that the single-stranded extension activity of Pol θ was the result of non-templated terminal transferase activity (). However, Pol θ is not a terminal transferase. Experiments in the study suggesting terminal transferase activity were conducted using Mn2+. This metal leads to promiscuous synthesis and a high rate of mismatch incorporation, and it is known that Pol θ can extend readily from a 3′-terminal mismatch (; ). Like other A family polymerases, Pol θ can add an additional dATP to a blunt DNA end, but otherwise shows predictable template requirements in a first round of DNA synthesis ().
DNA polymerase θ is the defining enzyme in a double strand break repair pathway, termed theta-mediated end-joining (TMEJ). During TMEJ, Pol θ engages single stranded 3′ ends by microhomology. It thus makes sense that Pol θ recognizes and processes ssDNA in several ways. Pol θ apparently manipulates the 3′ end of the ssDNA in the active site, forming a transitory hairpin that serves as the intermediate for either cleavage or extension of the 3′-end (). The factors that dictate the choice between these two activities are being investigated, as well as how processed single-strands are then advanced to a repair stage that uses pairing at microhomologies and primed extension on double-stranded DNA. Statistically, microhomologies will rarely be found at the ends of a double-strand break but will more often occur internal to the break (Figure 6A). The two steps of processing of DNA ends and templated extension appear to largely explain the mutational “signature” of TMEJ, involving short sequence insertions and deletions at the site of repair (; ; ).
Endonucleolytic cleavage of a polynucleotide chain is also a feature of RNA polymerases, which edit transcripts by cleaving off 1 or 2 nucleotides from the 3′ end (; ; ). This editing reactions are conducted in a Mg2+ - dependent manner from the RNA polymerase active site. The regulation of extension vs. cleavage is enhanced by protein cofactors that participate directly in reorganizing the amino acids in the catalytic site, referred to as “active center tuning” (). For example, the endonuclease activity of E. coli RNA polymerase is stimulated by Gre proteins, and eukaryotic RNA pol II by TFIIS. Stimulated by Gre, RNA polymerase clips off the two terminal nt of a synthetic RNA fragment (; ). In contrast to the pol θ end-trimming reaction, RNA polymerase editing does not seem to be directly dNTP- or NTP-dependent, although it can be modulated by nucleotide concentration. Other nucleotidyl transferases may also engage in a form of end-trimming or editing. For example, telomerase, including highly purified human telomerase, can shorten products as well as extend them (; ; ).
Activity on RNA
Some DNA polymerases perform nucleotidyl transferase reactions that do not involve the usual DNA substrates or primers, and are briefly mentioned here to further illustrate the enzymatic versatility of DNA polymerases.
Reverse Transcriptase
Reverse transcriptases are RNA-dependent DNA polymerases, which use an RNA template to synthesize DNA. Some DNA-dependent DNA polymerases have been reported to have reverse transcriptase activity. Two bacterial A-family polymerases, Bst DNA polymerase and the Klenow fragment of E. coli Pol I, were successfully used to reverse transcribe RNA in vitro (). Taq DNA polymerase can also engage in this activity, after mutating a single aspartate into asparagine (). Human pol η can use either a DNA duplex or and RNA/DNA mixed duplex during synthesis (; ), and was shown to function as a reverse transcriptase in a cellular environment (). Pol θ was recently reported to also have a reverse transcriptase activity, although additional studies are warranted to establish the physiological relevance of this finding ().
Primase-Polymerase
Some DNA polymerases, for example eukaryotic DNA pol α, have a separate primase subunit that allows RNA priming for DNA replication. PrimPol is the only known eukaryotic DNA polymerase with intrinsic priming activity. Both primase and DNA synthesis are mediated by the same active site. Recently it was found that a priming activity of DNA polymerase PrimPol is used for replication restart after DNA damage (; ). PrimPol is primarily a TLS polymerase which is found in both the nucleus and the mitochondria, and is capable of bypassing 7,8-dihydro-8-oxoguanine (oxoG) and cyclobutane pyrimidine dimers (CPD) (; ). PrimPol can synthesize short DNA primers which other polymerases, like mitochondrial polymerase γ, can use to restart replication promoting overall mitochondrial genome maintenance. Recent studies have found that PrimPol repriming activity is used by stalled replication forks to skip DNA lesions and restart replication, leaving behind ssDNA gaps that can be filled by other repair polymerases (; ).
Conclusion
DNA polymerases catalyze the nucleotidyl transfer reaction to generate a DNA polymer. In practice, however, this class of enzymes is able to catalyze a host of other DNA processing reactions that make DNA polymerases a multi-tool of genomic integrity. Redundancy in the activities of DNA polymerases is critical for replication and maintenance of the cell. If a DNA polymerase is unable to perform its function, other DNA polymerases are poised to intervene and assume the role of replication and repair typically filled by the compromised enzyme. These redundancies also facilitate the sheer amount of DNA replication and repair that must continuously occur in the cell throughout its life. It must be noted that the existence of redundant polymerase roles makes targeting DNA polymerases in cancer a challenge due to the ability of the cell to compensate for the loss by employing a different polymerase.
By using a DNA polymerase to execute more than one aspect of DNA processing, the cell can benefit energetically. Requiring a separate enzyme for each step would necessitate a greater energetic commitment from the cell to synthesize and recruit these enzymes than employing an existing polymerase. Furthermore, repurposing the active site of a DNA polymerase can facilitate spatial organization of enzymatic activities. By utilizing a common active site for multiple reactions, the polymerase becomes a hub where each activity may be performed sequentially. This arrangement is advantageous to the cell as a polymerase can process DNA in multiple ways before diffusing from the DNA, further minimizing the amount of energy required to attain appropriately processed DNA.
While DNA polymerases have been investigated for close to 60 years, we continue to discover noncanonical polymerase activities. From the addition of an enzymatic domain to exploiting an existing active site, DNA polymerases act like Swiss army knives of DNA processing. We can look forward to discovering what other functions these DNA multi-tools utilize to tackle DNA processing errors. Conveniently these multiple functions present additional opportunities to design small molecule inhibitors against these multitasking enzymes.
Statements
Author contributions
All authors contributed to planning of the article, writing, designing figures, and editing.
Funding
Work on DNA polymerases in our laboratories is funded by National Institutes of Health grant T32 AI055402 (LD), P01 CA247773 (SD and RW), and the J. Ralph Meadows Chair in Carcinogenesis Research (RW).
Acknowledgments
We acknowledge Prof. Tom Steitz, whose pioneering structural insights into nucleic acid polymerases made the work presented here possible.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
1
AllinsonS. L.DianovaI. I.DianovG. L. (2001). DNA Polymerase Beta Is the Major dRP Lyase Involved in Repair of Oxidative Base Lesions in DNA by Mammalian Cell Extracts. EMBO J.20 (23), 6919–6926. 10.1093/emboj/20.23.6919
2
BarnesW. M.ZhangZ.KermekchievM. B. (2021). A Single Amino Acid Change to Taq DNA Polymerase Enables Faster PCR, Reverse Transcription and Strand-Displacement. Front. Bioeng. Biotechnol.8. 10.3389/fbioe.2020.553474
3
BatraV. K.BeardW. A.ShockD. D.KrahnJ. M.PedersenL. C.WilsonS. H. (2006). Magnesium-induced Assembly of a Complete DNA Polymerase Catalytic Complex. Structure14 (4), 757–766. 10.1016/j.str.2006.01.011
4
BebenekK.TissierA.FrankE. G.McDonaldJ. P.PrasadR.WilsonS. H.et al (2001). 5'-Deoxyribose Phosphate Lyase Activity of Human DNA Polymerase Ɩ In Vitro. Science291(5511), 2156–2159. 10.1126/science.1058386
5
BeechemJ. M.OttoM. R.BloomL. B.EritjaR.Reha-KrantzL. J.GoodmanM. F. (1998). Exonuclease−Polymerase Active Site Partitioning of Primer−Template DNA Strands and Equilibrium Mg2+ Binding Properties of Bacteriophage T4 DNA Polymerase. Biochemistry37 (28), 10144–10155. 10.1021/bi980074b
6
BeeseL. S.DerbyshireV.SteitzT. A. (1993). Structure of DNA Polymerase I Klenow Fragment Bound to Duplex DNA. Science260 (5106), 352–355. 10.1126/science.8469987
7
BernadA.BlancoL.LázaroJ.MartínG.SalasM. (1989). A Conserved 3′→5′ Exonuclease Active Site in Prokaryotic and Eukaryotic DNA Polymerases. Cell59 (1), 219–228. 10.1016/0092-8674(89)90883-0
8
BiberS.PospiechH.GottifrediV.WiesmüllerL. (2020). Multiple Biochemical Properties of the P53 Molecule Contribute to Activation of Polymerase iota-dependent DNA Damage Tolerance. Nucleic Acids Res.48 (21), 12188–12203. 10.1093/nar/gkaa974
9
BorukhovS.SagitovV.GoldfarbA. (1993). Transcript Cleavage Factors from E. coli. Cell72 (3), 459–466. 10.1016/0092-8674(93)90121-6
10
Carvajal-GarciaJ.ChoJ.-E.Carvajal-GarciaP.FengW.WoodR. D.SekelskyJ.et al (2020). Mechanistic Basis for Microhomology Identification and Genome Scarring by Polymerase Theta. Proc. Natl. Acad. Sci. USA117 (15), 8476–8485. 10.1073/pnas.1921791117
11
CeskaT.SayersJ. R. (1998). Structure-specific DNA Cleavage by 5′ Nucleases. Trends Biochem. Sci.23 (9), 331–336. 10.1016/s0968-0004(98)01259-6
12
ChandramoulyG.ZhaoJ.McDevittS.RusanovT.HoangT.BorisonnikN.et al (2021). Pol θ Reverse Transcribes RNA and Promotes RNA-Templated DNA Repair. Sci. Adv.7, eabf1771.
13
CollinsK.GreiderC. W. (1993). Tetrahymena Telomerase Catalyzes Nucleolytic Cleavage and Nonprocessive Elongation. Genes Develop.7 (7b), 1364–1376. 10.1101/gad.7.7b.1364
14
DalalS.ChikovaA.JaegerJ.SweasyJ. B. (2008). The Leu22Pro Tumor-Associated Variant of DNA Polymerase Beta Is dRP Lyase Deficient. Nucleic Acids Res.36 (2), 411–422. 10.1093/nar/gkm1053
15
Del PradoA.Franco-EchevarríaE.GonzálezB.BlancoL.SalasM.de VegaM. (2018). Noncatalytic Aspartate at the Exonuclease Domain of Proofreading DNA Polymerases Regulates Both Degradative and Synthetic Activities. Proc. Natl. Acad. Sci. USA115 (13), E2921–E2929. 10.1073/pnas.1718787115
16
DerbyshireV.FreemontP. S.SandersonM. R.BeeseL.FriedmanJ. M.JoyceC. M.et al (1988). Genetic and Crystallographic Studies of the 3′,5′-Exonucleolytic Site of DNA Polymerase I. Science240 (4849), 199–201. 10.1126/science.2832946
17
DoubliéS.TaborS.LongA. M.RichardsonC. C.EllenbergerT. (1998). Crystal Structure of a Bacteriophage T7 DNA Replication Complex at 2.2 Å Resolution. Nature391 (6664), 251–258. 10.1038/34593
18
DoubliéS.ZahnK. E. (2014). Structural Insights into Eukaryotic DNA Replication. Front. Microbiol.5, 444. 10.3389/fmicb.2014.00444
19
FiléeJ.ForterreP.Sen-LinT.LaurentJ. (2002). Evolution of DNA Polymerase Families: Evidences for Multiple Gene Exchange between Cellular and Viral Proteins. J. Mol. Evol.54 (6), 763–773. 10.1007/s00239-001-0078-x
20
FortiniP.PascucciB.ParlantiE.SobolR. W.WilsonS. H.DogliottiE. (1998). Different DNA Polymerases Are Involved in the Short- and Long-Patch Base Excision Repair in Mammalian Cells. Biochemistry37 (11), 3575–3580. 10.1021/bi972999h
21
FouquerelE.LormandJ.BoseA.LeeH.-T.KimG. S.LiJ.et al (2016). Oxidative Guanine Base Damage Regulates Human Telomerase Activity. Nat. Struct. Mol. Biol.23 (12), 1092–1100. 10.1038/nsmb.3319
22
FranklinA.MilburnP. J.BlandenR. V.SteeleE. J. (2004). Human DNA Polymerase-η, an A-T Mutator in Somatic Hypermutation of Rearranged Immunoglobulin Genes, is a Reverse Transcriptase. Immunol. Cell Biol.82, 219–225.
23
FranklinM. C.WangJ.SteitzT. A. (2001). Structure of the Replicating Complex of a Pol α Family DNA Polymerase. Cell105 (5), 657–667. 10.1016/s0092-8674(01)00367-1
24
GammonD. B.EvansD. H. (2009). The 3′-to-5′ Exonuclease Activity of Vaccinia Virus DNA Polymerase Is Essential and Plays a Role in Promoting Virus Genetic Recombination. J. Virol.83 (9), 4236–4250. 10.1128/jvi.02255-08
25
GanaiR. A.BylundG. O.JohanssonE. (2015). Switching between Polymerase and Exonuclease Sites in DNA Polymerase ε. Nucleic Acids Res.43 (2), 932–942. 10.1093/nar/gku1353
26
Garcia-GomezS.ReyesA.Martinez-JimenezM. I.ChocronE. S.MouronS.TerradosG.et al (2013). PrimPol, an Archaic Primase/Polymerase Operating in Human Cells. Mol. Cell52, 541–553.
27
GrasbyJ. A.FingerL. D.TsutakawaS. E.AtackJ. M.TainerJ. A. (2012). Unpairing and Gating: Sequence-independent Substrate Recognition by FEN Superfamily Nucleases. Trends Biochem. Sci.37 (2), 74–84. 10.1016/j.tibs.2011.10.003
28
GutmanP. D.MintonK. W. (1993). Conserved Sites in the 5'-3' Exonuclease Domain of Escherichia coli DNA Polymerase. Nucl. Acids Res.21 (18), 4406–4407. 10.1093/nar/21.18.4406
29
HamppS.KiesslingT.BuechleK.MansillaS. F.ThomaleJ.RallM.et al (2016). DNA Damage Tolerance Pathway Involving DNA Polymerase ι and the Tumor Suppressor P53 Regulates DNA Replication fork Progression. Proc. Natl. Acad. Sci. USA113 (13), E4311–E4319. 10.1073/pnas.1605828113
30
HarringtonJ. J.LieberM. R. (1994). The Characterization of a Mammalian DNA Structure-specific Endonuclease. EMBO J.13 (5), 1235–1246. 10.1002/j.1460-2075.1994.tb06373.x
31
HeP.YangW. (2018). Template and Primer Requirements for DNA Pol θ-mediated End Joining. Proc. Natl. Acad. Sci. USA115 (30), 7747–7752. 10.1073/pnas.1807329115
32
HoggM.AllerP.KonigsbergW.WallaceS. S.DoubliéS. (2007). Structural and Biochemical Investigation of the Role in Proofreading of a β Hairpin Loop Found in the Exonuclease Domain of a Replicative DNA Polymerase of the B Family. J. Biol. Chem.282 (2), 1432–1444. 10.1074/jbc.m605675200
33
HoggM.Sauer-ErikssonA. E.JohanssonE. (2012). Promiscuous DNA Synthesis by Human DNA Polymerase θ. Nucleic Acids Res.40(6), 2611–2622. 10.1093/nar/gkr1102
34
HoggM.WallaceS. S.DoubliéS. (2004). Crystallographic Snapshots of a Replicative DNA Polymerase Encountering an Abasic Site. EMBO J.23 (7), 1483–1493. 10.1038/sj.emboj.7600150
35
HowardM. J.HortonJ. K.ZhaoM.-L.WilsonS. H. (2020). Lysines in the Lyase Active Site of DNA Polymerase β Destabilize Nonspecific DNA Binding, Facilitating Searching and DNA gap Recognition. J. Biol. Chem.295 (34), 12181–12187. 10.1074/jbc.ra120.013547
36
HwangT.RehS.DunbayevY.ZhongY.TakataY.ShenJ.et al (2020). Defining the Mutation Signatures of DNA Polymerase θ in Cancer Genomes. NAR Cancer2 (3), zcaa017. 10.1093/narcan/zcaa017
37
IzbanM. G.LuseD. S. (1992). The RNA Polymerase II Ternary Complex Cleaves the Nascent Transcript in a 3'----5' Direction in the Presence of Elongation Factor SII. Genes Dev.6 (7), 1342–1356. 10.1101/gad.6.7.1342
38
JoyceC. M. (2013). “DNA Polymerase I, Bacterial,” in Encyclopedia of Biological Chemistry. Editors LennarzW.J.LaneM.D.. Second Edition (Waltham: Academic Press), 87–91. 10.1016/b978-0-12-378630-2.00306-6
39
JoyceC. M.SunX. C.GrindleyN. D. (1992). Reactions at the Polymerase Active Site that Contribute to the Fidelity of Escherichia coli DNA Polymerase I (Klenow Fragment). J. Biol. Chem.267 (34), 24485–24500. 10.1016/s0021-9258(18)35792-2
40
KentT.Mateos-GomezP. A.SfeirA.PomerantzR. T. (2016). Polymerase θ Is a Robust Terminal Transferase that Oscillates between Three Different Mechanisms during End-Joining. eLife5, e13740. 10.7554/eLife.13740
41
KhareV.EckertK. A. (2002). The Proofreading 3′→5′ Exonuclease Activity of DNA Polymerases: a Kinetic Barrier to Translesion DNA Synthesis. Mutat. Research/Fundamental Mol. Mech. Mutagenesis510 (1–2), 45–54. 10.1016/s0027-5107(02)00251-8
42
KhodyrevaS. N.PrasadR.IlinaE. S.SukhanovaM. V.KutuzovM. M.LiuY.et al (2010). Apurinic/apyrimidinic (AP) Site Recognition by the 5'-dRP/AP Lyase in poly(ADP-Ribose) Polymerase-1 (PARP-1). Proc. Natl. Acad. Sci.107 (51), 22090–22095. 10.1073/pnas.1009182107
43
KimY.EomS. H.WangJ.LeeD.-S.SuhS. W.SteitzT. A. (1995). Crystal Structure of Thermus Aquaticus DNA Polymerase. Nature376(6541), 612–616. 10.1038/376612a0
44
KlenowH.Overgaard-HansenK. (1970). Proteolytic Cleavage of DNA Polymerase from Escherichia Coli B into an Exonuclease Unit and a Polymerase Unit. FEBS Lett.6 (1), 25–27. 10.1016/0014-5793(70)80032-1
45
KlunglandA.LindahlT. (1997). Second Pathway for Completion of Human DNA Base Excision-Repair: Reconstitution with Purified Proteins and Requirement for DNase IV (FEN1). EMBO J.16 (11), 3341–3348. 10.1093/emboj/16.11.3341
46
LaiY.WeizmannY.LiuY. (2018). The Deoxyribose Phosphate Lyase of DNA Polymerase β Suppresses a Processive DNA Synthesis to Prevent Trinucleotide Repeat Instability. Nucleic Acids Res.46 (17), 8940–8952. 10.1093/nar/gky700
47
LamichhaneR.BerezhnaS. Y.GillJ. P.Van der SchansE.MillarD. P. (2013). Dynamics of Site Switching in DNA Polymerase. J. Am. Chem. Soc.135 (12), 4735–4742. 10.1021/ja311641b
48
LavertyD. J.GreenbergM. M. (2018). Expanded Substrate Scope of DNA Polymerase θ and DNA Polymerase β: Lyase Activity on 5′-Overhangs and Clustered Lesions. Biochemistry57 (42), 6119–6127. 10.1021/acs.biochem.8b00911
49
LavertyD. J.MortimerI. P.GreenbergM. M. (2018). Mechanistic Insight through Irreversible Inhibition: DNA Polymerase θ Uses a Common Active Site for Polymerase and Lyase Activities. J. Am. Chem. Soc.140 (29), 9034–9037. 10.1021/jacs.8b04158
50
LiY.DuttaS.DoubliéS.BdourH. M. d.TaylorJ.-S.EllenbergerT. (2004). Nucleotide Insertion Opposite a Cis-Syn Thymine Dimer by a Replicative DNA Polymerase from Bacteriophage T7. Nat. Struct. Mol. Biol.11 (8), 784–790. 10.1038/nsmb792
51
LongleyM. J.BennettS. E.MosbaughD. W. (1990). Characterization of the 5′ to 3′ Exonuclease Associated with Thermus aquaticus DNA Polymerase. Nucl. Acids Res.18 (24), 7317–7322. 10.1093/nar/18.24.7317
52
LyamichevV.BrowM. A. D.VarvelV. E.DahlbergJ. E. (1999). Comparison of the 5' Nuclease Activities of Taq DNA Polymerase and its Isolated Nuclease Domain. Proc. Natl. Acad. Sci.96 (11), 6143–6148. 10.1073/pnas.96.11.6143
53
MatsumotoY.KimK. (1995). Excision of Deoxyribose Phosphate Residues by DNA Polymerase β during DNA Repair. Science269 (24), 699–702. 10.1126/science.7624801
54
MouronS.Rodriguez-AcebesS.Martinez-JimenezM. I.Garcia-GomezS.ChocronS.BlancoL.et al (2013). Repriming of DNA Synthesis at Stalled Replication Forks by Human PrimPol. Nat. Struct. Mol. Biol.20, 1383–1389.
55
ParkY.ChoiH.LeeD. S.KimY. (1997). Improvement of the 3'-5' Exonuclease Activity of Taq DNA Polymerase by Protein Engineering in the Active Site. Mol. Cells7 (3), 419–424.
56
PatelP. H.SuzukiM.AdmanE.ShinkaiA.LoebL. A. (2001). Prokaryotic DNA Polymerase I: Evolution, Structure, and "base Flipping" Mechanism for Nucleotide Selection. J. Mol. Biol.308 (5), 823–837. 10.1006/jmbi.2001.4619
57
PauszekR. F.IIILamichhaneR.Rajkarnikar SinghA.MillarD. P. (2021). Single-molecule View of Coordination in a Multi-Functional DNA Polymerase. eLife10, e62046. 10.7554/elife.62046
58
PellicanòG.Al MamunM.Jurado-SantiagoD.Villa-HernándezS.YinX.GiannattasioM.et al (2021). Checkpoint-mediated DNA Polymerase ε Exonuclease Activity Curbing Counteracts Resection-Driven fork Collapse. Mol. Cell81 (13), 2778–e4. 10.1016/j.molcel.2021.04.006
59
PettaT. B.NakajimaS.ZlatanouA.DesprasE.Couve-PrivatS.IshchenkoA.et al (2008). Human DNA Polymerase iota Protects Cells against Oxidative Stress. EMBO J.27 (21), 2883–2895. 10.1038/emboj.2008.210
60
PinzK. G.BogenhagenD. F. (2000). Characterization of a Catalytically Slow AP Lyase Activity in DNA Polymerase γ and Other Family A DNA Polymerases. J. Biol. Chem.275 (17), 12509–12514. 10.1074/jbc.275.17.12509
61
PrasadR.BeardW. A.ChyanJ. Y.MaciejewskiM. W.MullenG. P.WilsonS. H. (1998). Functional Analysis of the Amino-Terminal 8-kDa Domain of DNA Polymerase β as Revealed by Site-Directed Mutagenesis. J. Biol. Chem.273(18), 11121–11126. 10.1074/jbc.273.18.11121
62
PrasadR.BeardW. A.WilsonS. H. (1994). Studies of Gapped DNA Substrate Binding by Mammalian DNA Polymerase Beta. Dependence on 5'-phosphate Group. J. Biol. Chem.269 (27), 18096–18101. 10.1016/s0021-9258(17)32422-5
63
PrasadR.BebenekK.HouE.ShockD. D.BeardW. A.WoodgateR.et al (2003). Localization of the Deoxyribose Phosphate Lyase Active Site in Human DNA Polymerase ι by Controlled Proteolysis. J. Biol. Chem.278 (32), 29649–29654. 10.1074/jbc.m305399200
64
PrasadR.HortonJ. K.ChastainP. D.GassmanN. R.FreudenthalB. D.HouE. W.et al (2014). Suicidal Cross-Linking of PARP-1 to AP Site Intermediates in Cells Undergoing Base Excision Repair. Nucleic Acids Res.42 (10), 6337–6351. 10.1093/nar/gku288
65
PrasadR.LongleyM. J.ShariefF. S.HouE. W.CopelandW. C.WilsonS. H. (2009). Human DNA Polymerase Possesses 5'-dRP Lyase Activity and Functions in Single-Nucleotide Base Excision Repair In Vitro. Nucleic Acids Res.37 (6), 1868–1877. 10.1093/nar/gkp035
66
QuinetA.TirmanS.JacksonJ.ŠvikovićS.LemaconD.Carvajal-MaldonadoD.et al (2020). PRIMPOL-Mediated Adaptive Response Suppresses Replication Fork Reversal in BRCA-Deficient Cells. Mol. Cell77, 461–474.
67
RechkoblitO.JohnsonR. E.GuptaY. K.PrakashL.PrakashS.AggarwalA. K. (2021). Structural Basis of DNA Synthesis Opposite 8-Oxoguanine by Human PrimPol Primase-Polymerase. Nat. Commun.12, 4020.
68
ReinesD. (1992). Elongation Factor-dependent Transcript Shortening by Template-Engaged RNA Polymerase II. J. Biol. Chem.267 (6), 3795–3800. 10.1016/s0021-9258(19)50596-8
69
RobinsP.PappinD. J.WoodR. D.LindahlT. (1994). Structural and Functional Homology between Mammalian DNase IV and the 5'-nuclease Domain of Escherichia coli DNA Polymerase I. J. Biol. Chem.269 (46) 28535–28538. 10.1016/s0021-9258(19)61935-6
70
RubtsovaM. P.VasilkovaD. P.MalyavkoA. N.NaraikinaY. V.ZverevaM. I.DontsovaO. A. (2012). Telomere Lengthening and Other Functions of Telomerase. Acta Naturae4 (2), 44–61. 10.32607/actanaturae.10630
71
SchimmelJ.van SchendelR.den DunnenJ. T.TijstermanM. (2019). Templated Insertions: A Smoking Gun for Polymerase Theta-Mediated End Joining. Trends Genet.35 (9), 632–644. 10.1016/j.tig.2019.06.001
72
SekiM.WoodR. D. (2008). DNA Polymerase θ (POLQ) Can Extend from Mismatches and from Bases Opposite a (6-4) Photoproduct. DNA Repair7 (1), 119–127. 10.1016/j.dnarep.2007.08.005
73
ShanbhagV.SachdevS.FloresJ.ModakM.SinghK. (2018). Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions. Biology7 (1), 5. 10.3390/biology7010005
74
ShevelevI. V.HübscherU. (2002). The 3′-5′ Exonucleases. Nat. Rev. Mol. Cel Biol3 (5), 364–376. 10.1038/nrm804
75
ShiC.ShenX.NiuS.MaC. (2015). Innate Reverse Transcriptase Activity of DNA Polymerase for Isothermal RNA Direct Detection. J. Am. Chem. Soc.137, 13804–13806.
76
SobolR. W.HortonJ. K.KühnR.GuH.SinghalR. K.PrasadR.et al (1996). Requirement of Mammalian DNA Polymerase-β in Base-Excision Repair. Nature379 (6561), 183–186. 10.1038/379183a0
77
SobolR. W.PrasadR.EvenskiA.BakerA.YangX.-P.HortonJ. K.et al (2000). The Lyase Activity of the DNA Repair Protein β-polymerase Protects from DNA-Damage-Induced Cytotoxicity. Nature405 (6788), 807–810. 10.1038/35015598
78
SosunovV.SosunovaE.MustaevA.BassI.NikiforovV.GoldfarbA. (2003). Unified Two-Metal Mechanism of RNA Synthesis and Degradation by RNA Polymerase. EMBO J.22 (9), 2234–2244. 10.1093/emboj/cdg193
79
SosunovaE.SosunovV.EpshteinV.NikiforovV.MustaevA. (2013). Control of Transcriptional Fidelity by Active Center Tuning as Derived from RNA Polymerase Endonuclease Reaction. J. Biol. Chem.288 (9), 6688–6703. 10.1074/jbc.m112.424002
80
SteitzT. A. (1999). DNA Polymerases: Structural Diversity and Common Mechanisms. J. Biol. Chem.274 (25), 17395–17398. 10.1074/jbc.274.25.17395
81
SuY.EgliM.GuengerichF. P. (2017). Human DNA Polymerase eta Accommodates RNA for Strand Extension. J. Biol. Chem.292, 18044–18051.
82
SuY.GhodkeP. P.EgliM.LiL.WangY.GuengerichF. P. (2019). Human DNA Polymerase η has Reverse Transcriptase Activity in Cellular Environments. J. Biol. Chem.294, 6073–6081.
83
TirmanS.QuinetA.WoodM.MeroniA.CybullaE.JacksonJ.et al (2021). Temporally Distinct Post-Replicative Repair Mechanisms Fill PRIMPOL-Dependent ssDNA Gaps in Human Cells. Mol. Cell81, 4026–4040.
84
TsutakawaS. E.ClassenS.ChapadosB. R.ArvaiA. S.FingerL. D.GuentherG.et al (2011). Human Flap Endonuclease Structures, DNA Double-Base Flipping, and a Unified Understanding of the FEN1 Superfamily. Cell145 (2), 198–211. 10.1016/j.cell.2011.03.004
85
WangR.LenoirW. F.WangC.SuD.McLaughlinM.HuQ.et al (2020). DNA Polymerase ι Compensates for Fanconi Anemia Pathway Deficiency by Countering DNA Replication Stress. Proc. Natl. Acad. Sci. USA117 (52), 33436–33445. 10.1073/pnas.2008821117
86
XuY.GrindleyN. D. F.JoyceC. M. (2000). Coordination between the Polymerase and 5′-Nuclease Components of DNA Polymerase I of Escherichia coli. J. Biol. Chem.275 (27), 20949–20955. 10.1074/jbc.m909135199
87
YangW.LeeJ. Y.NowotnyM. (2006). Making and Breaking Nucleic Acids: Two-Mg2+-Ion Catalysis and Substrate Specificity. Mol. Cel22 (1), 5–13. 10.1016/j.molcel.2006.03.013
88
YousefzadehM. J.WyattD. W.TakataK.-I.MuY.HensleyS. C.TomidaJ.et al (2014). Mechanism of Suppression of Chromosomal Instability by DNA Polymerase POLQ. Plos Genet.10 (10), e1004654. 10.1371/journal.pgen.1004654
89
ZahnK. E.AverillA. M.AllerP.WoodR. D.DoubliéS. (2015). Human DNA Polymerase θ Grasps the Primer Terminus to Mediate DNA Repair. Nat. Struct. Mol. Biol.22 (4), 304–311. 10.1038/nsmb.2993
90
ZahnK. E.JensenR. B.WoodR. D.DoubliéS. (2021). Human DNA Polymerase θ Harbors DNA End-Trimming Activity Critical for DNA Repair. Mol. Cel81 (7), 1534–1547. 10.1016/j.molcel.2021.01.021
91
ZuoY.DeutscherM. P. (2002). The Physiological Role of RNase T Can Be Explained by its Unusual Substrate Specificity. J. Biol. Chem.277 (33), 29654–29661. 10.1074/jbc.m204252200
Summary
Keywords
DNA polymerases, nucleotidyl transfer, DNA repair, nuclease activity, lyase activity, proofreading
Citation
Carvajal-Maldonado D, Drogalis Beckham L, Wood RD and Doublié S (2022) When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer. Front. Mol. Biosci. 8:815845. doi: 10.3389/fmolb.2021.815845
Received
15 November 2021
Accepted
13 December 2021
Published
07 January 2022
Volume
8 - 2021
Edited by
Janice Pata, Wadsworth Center, United States
Reviewed by
Todd Washington, The University of Iowa, United States
Linlin Zhao, University of California, Riverside, United States
Updates
Copyright
© 2022 Carvajal-Maldonado, Drogalis Beckham, Wood and Doublié.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Sylvie Doublié, sdoublie@uvm.edu; Richard D. Wood, rwood@mdanderson.org
This article was submitted to Structural Biology, a section of the journal Frontiers in Molecular Biosciences
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.