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Exonuclease

An exonuclease is an enzyme that catalyzes the sequential removal of nucleotide monophosphates from the 3' or 5' ends of DNA or RNA strands through hydrolysis of phosphodiester bonds.^[1] These enzymes play essential roles in nucleic acid metabolism, distinguishing themselves from endonucleases by their specific action at chain termini rather than internal sites.^[1] Exonucleases are classified by directionality: 5' to 3' exonucleases, such as EXO1 and FEN1, degrade strands starting from the 5' end, while 3' to 5' exonucleases, including TREX1 and the proofreading domains of DNA polymerases like Pol δ and Pol ε, proceed from the 3' end.^[2] This polarity determines their involvement in specific processes, with 3' to 5' activity often linked to error correction during synthesis and 5' to 3' to flap processing or resection.^[1] In cellular biology, exonucleases are critical for maintaining genome integrity through proofreading during DNA replication to excise mismatched bases, removing RNA primers from Okazaki fragments, and processing DNA ends in recombination and telomere maintenance.^[2] They participate in multiple DNA repair pathways, including base excision repair (BER) via APE1's activity, nucleotide excision repair (NER), mismatch repair (MMR) primarily through EXO1, and double-strand break repair mechanisms like homologous recombination (HR) and non-homologous end joining (NHEJ) involving enzymes such as MRE11 and Artemis.^[1] Beyond repair, exonucleases regulate immune responses by degrading cytosolic DNA or RNA to prevent inflammatory activation, as seen with TREX1 in averting autoimmunity, and facilitate programmed cell death by fragmenting chromatin during apoptosis.^[2] Dysfunctions in these enzymes are associated with genomic instability, premature aging syndromes like Werner syndrome (due to WRN mutations), cancer predisposition, and autoimmune disorders such as Aicardi-Goutières syndrome.^[1]

General Properties

Definition and Function

An exonuclease is an enzyme that catalyzes the hydrolysis of phosphodiester bonds at the 3' or 5' termini of DNA or RNA strands, removing nucleotides sequentially in either a processive or distributive manner.^[3]^[4] This exonucleolytic activity distinguishes it from other nucleases by targeting the ends of polynucleotide chains rather than internal sites.^[5] Exonucleases are classified within the hydrolase class (EC 3.1), with specific subgroups such as EC 3.1.11 for exodeoxyribonucleases producing 5'-phosphomonoesters and EC 3.1.13 for exoribonucleases acting on RNA.^[6] These enzymes play essential roles in nucleic acid metabolism across all domains of life.^[7] The primary functions of exonucleases include the degradation of nucleic acids to recycle nucleotides or eliminate damaged strands, proofreading during DNA replication to excise mismatched bases and enhance fidelity, maturation of RNA by trimming precursors to functional forms, and maintenance of telomeres by processing chromosome ends to prevent genomic instability.^[2] For instance, in DNA replication, exonucleases associated with polymerases remove errors, reducing mutation rates significantly.^[2] In RNA processing, they contribute to the turnover of transcripts, ensuring proper gene expression regulation.^[8] These activities underscore the broad applications of exonucleases in cellular quality control, where they safeguard genome stability by repairing or degrading aberrant nucleic acids and facilitate RNA turnover to maintain homeostasis.^[1] Deficiencies in exonuclease function are linked to diseases such as cancer and autoimmune disorders due to accumulated genomic damage.^[2]

Distinction from Other Nucleases

Exonucleases are distinguished from endonucleases by their mode of action on nucleic acids, as exonucleases sequentially hydrolyze phosphodiester bonds starting from the 5′ or 3′ termini of DNA or RNA strands, progressively releasing nucleotides one at a time, whereas endonucleases cleave internal phosphodiester bonds to generate oligonucleotide fragments without requiring free ends.^[5] This end-specific activity enables exonucleases to process terminal sequences in processes like degradation or maturation, in contrast to the sequence-specific or nonspecific internal cuts performed by endonucleases such as restriction enzymes.^[9] Unlike phosphatases, which catalyze the hydrolysis of phosphomonoester bonds to release inorganic phosphate from substrates like nucleotide monophosphates or phosphorylated proteins, exonucleases target phosphodiester bonds in polynucleotides and release nucleoside monophosphates as products, retaining the nucleotide base, sugar, and phosphate in a single unit. This distinction arises from the substrate specificity: phosphatases act on terminal phosphate groups, while exonucleases degrade the polymer backbone to yield mononucleotide byproducts.^[5] Structurally, exonucleases frequently possess specialized domains that facilitate binding to single-stranded DNA or RNA termini, such as the helical arch in T5 5′-exonuclease or the narrow channel in RNase II that accommodates end-on substrate entry, allowing processive degradation from exposed ends.^[5] In human exonuclease 1 (Exo1), for instance, the enzyme binds gapped or nicked DNA by bending the substrate and threading single-stranded segments into the active site, a feature adapted for terminal processing.00242-X) These binding elements contrast with the more varied active site geometries in endonucleases, which often lack such terminus-specific grips. Evolutionarily, exonucleases represent a specialization within the broader nuclease superfamily for terminal nucleic acid processing, diverging from endonucleases that evolved for internal cleavage roles in defense and recombination; both share ancient two-metal-ion catalytic cores but exhibit convergent adaptations in folds like the DnaQ motif for exonucleases versus RNase H-like structures for many endonucleases.^[5] This divergence underscores their complementary functions in nucleic acid metabolism, with exonucleases optimizing end resection and proofreading across prokaryotes and eukaryotes.^[10]

Biochemical Mechanism

Cleavage Process

Exonucleases catalyze the hydrolysis of terminal phosphodiester bonds in nucleic acids, initiating a nucleophilic attack by a water molecule on the phosphorus atom of the scissile bond. This reaction typically proceeds via an S<sub>N</sub>2 mechanism, forming a transient penta-covalent intermediate at the phosphate, and often inverts the stereochemistry at the phosphorus. Divalent metal ions, such as Mg²⁺ or Mn²⁺, play a crucial role by coordinating the non-bridging oxygen atoms of the phosphate and activating the nucleophilic water through deprotonation, often via a two-metal-ion mechanism where the ions are positioned approximately 4 Å apart to stabilize the transition state.^[5] The cleavage process occurs in a stepwise manner, beginning with the binding of the exonuclease to the terminus of the nucleic acid substrate, which positions the terminal phosphodiester bond within the enzyme's active site. Conserved residues, such as aspartate or histidine, along with the metal ions, facilitate the alignment of the substrate and the incoming water molecule. Hydrolysis then cleaves the bond, releasing a single nucleotide monophosphate—typically as a 5'-monophosphate with a 3'-OH group on the shortened polynucleotide chain—and allowing the enzyme-substrate complex to adjust for the next cycle if processive. This release step ensures the progressive shortening of the nucleic acid strand without internal damage.^[5] Exonucleases exhibit two primary kinetic modes: processive, where the enzyme remains bound to the substrate and performs multiple sequential cleavages without dissociation, and distributive, where the enzyme dissociates after each nucleotide removal, requiring rebinding for subsequent cycles. Processivity is often enabled by structural features like narrow channels that thread the nucleic acid, as seen in enzymes such as λ-exonuclease, while distributive behavior predominates in others like Exonuclease III, influencing their efficiency in degrading substrates of varying lengths.^[5]^[11] The general reaction can be represented as:

(\ce{DNA})_n + \ce{H2O} \rightarrow (\ce{DNA})_{n-1} + \ce{dNMP}

where dNMP denotes a deoxyribonucleoside 5'-monophosphate.^[11]

Directionality and Specificity

Exonucleases are classified primarily by their directionality of nucleotide removal from the termini of nucleic acid strands, either in the 3' to 5' or 5' to 3' orientation.^[5] Those with 3' to 5' polarity initiate degradation at the 3' hydroxyl end and proceed toward the 5' phosphate end, a mechanism that allows for precise removal of nucleotides in a direction opposite to the typical 5' to 3' synthesis of nucleic acids, facilitating error correction and quality control processes.^[5] This directionality is exemplified by enzymes such as TREX1 and WRN, which exhibit high processivity on appropriate substrates.^[2] In contrast, 5' to 3' exonucleases begin cleavage at the 5' phosphate end and move toward the 3' hydroxyl end, enabling the coordinated excision of segments such as RNA primers or displaced strands during nucleic acid maturation.^[5] Enzymes like EXO1 and FEN1 demonstrate this polarity, often processing structured intermediates with efficiency that supports downstream metabolic pathways.^[2] Specificity in exonucleases is determined by substrate preferences, including a strong bias toward single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA), as the latter's helical stability hinders access to the phosphodiester backbone.^[5] For instance, many 3' to 5' exonucleases, such as those in the DnaQ family, preferentially degrade ssDNA, while some 5' to 3' variants like RecJ target ssDNA ends but can extend to dsDNA under specific conditions.^[5] Additionally, secondary structures like hairpins or loops can inhibit processive degradation by impeding enzyme translocation, requiring accessory factors to unwind or bypass these obstacles for continued activity.^[2] A representative example of directionality and specificity is Exonuclease I, a 3' to 5' enzyme that exclusively targets ssDNA, processively releasing deoxyribonucleoside 5'-monophosphates without affecting dsDNA or RNA, thereby highlighting its role in selective strand degradation.^[12]

Role in DNA Replication and Repair

Proofreading in Polymerases

In DNA replication, proofreading by exonucleases integrated into polymerase holoenzymes plays a crucial role in maintaining genomic fidelity by excising mismatched nucleotides immediately after incorporation. Replicative DNA polymerases, such as those in prokaryotic and eukaryotic systems, possess an intrinsic 3′→5′ exonuclease activity that detects and removes incorrectly paired bases during synthesis.^[13] This mechanism operates through a dynamic switching process: upon incorporation of a mismatched nucleotide, the polymerase stalls due to reduced extension efficiency, allowing the primer terminus to translocate from the polymerase active site to the adjacent exonuclease site for hydrolysis.^[14] The mispaired nucleotide is then excised, and the DNA shifts back to the polymerase site to resume accurate synthesis, ensuring minimal propagation of errors.^[14] The structural basis for this proofreading function lies in the exonuclease domain, which is covalently linked to the polymerase core within the holoenzyme complex. In bacterial DNA polymerase III, for example, this domain is provided by the epsilon subunit, which coordinates with the alpha subunit's polymerase activity to form a compact unit capable of rapid error correction. Similar architectures are observed in eukaryotic polymerases δ and ε, where the exonuclease motifs are embedded in the catalytic subunits, facilitating efficient substrate transfer without dissociation of the DNA.^[15] This intimate association minimizes the time required for proofreading, enhancing the overall speed and accuracy of replication. The significance of this exonuclease-mediated proofreading is evident in its substantial reduction of replication errors; without it, the intrinsic error rate of nucleotide selection alone is approximately 10^{-5} errors per base pair, but proofreading lowers this to about 10^{-7}, providing a 100-fold improvement in fidelity. This correction step is essential for preventing mutations that could lead to cellular dysfunction or disease, underscoring the evolutionary conservation of proofreading domains across diverse organisms.^[16]

Involvement in DNA Repair Pathways

Exonucleases are integral to multiple DNA repair pathways, where they resect damaged strands to remove lesions, process repair intermediates, and generate substrates for downstream repair mechanisms such as ligation or recombination. These enzymes exhibit direction-specific activities—typically 5'→3' or 3'→5'—that ensure precise excision without excessive degradation of healthy DNA. Their roles extend beyond replication proofreading to post-damage repair, contributing to genomic stability across prokaryotes and eukaryotes. In nucleotide excision repair (NER), which addresses bulky DNA lesions like UV-induced photoproducts, 5'→3' exonucleases facilitate strand resection following initial incisions. The human XPG protein, homologous to yeast RAD2, possesses a conserved 5'→3' exonuclease activity that supports the incision step by processing single- and double-stranded DNA regions near the damage site.^[17] Additionally, human exonuclease 1 (EXO1) localizes to UV-irradiated sites in a manner dependent on the NER preincision complex and XPF endonuclease activity, where it promotes unscheduled DNA synthesis and links NER to checkpoint activation via enhanced H2A ubiquitylation and Chk1 phosphorylation.^[18] In mismatch repair (MMR), EXO1 plays a central role by performing 5'→3' exonuclease activity to excise the newly synthesized DNA strand containing the mismatch, starting from a pre-existing nick or break directed by MutSα/MutLα recognition. This resection generates a gap that is subsequently filled by DNA polymerase, ensuring correction of replication errors and maintenance of genomic stability. EXO1's activity in MMR is essential, as its deficiency leads to microsatellite instability and cancer predisposition.^[19] Base excision repair (BER) relies on exonucleases to handle small, non-bulky lesions such as uracil or abasic (AP) sites, often through 3'→5' activities that clean repair intermediates. In Escherichia coli, exonuclease III (ExoIII, encoded by xth) is essential for repairing uracil-DNA glycosylase-generated AP sites in deoxyuridine triphosphatase-deficient mutants, exhibiting endonucleolytic nicking followed by exonucleolytic removal to prevent lethal accumulation of unrepaired sites.^[20] In humans, apurinic endonuclease 1 (APE1) displays 3'→5' exonuclease activity that preferentially processes mismatched 3' termini at nicks and one-nucleotide gaps, acting as a proofreading mechanism after DNA synthesis; this activity is inhibited by proximal 5'-incised AP residues but relieved upon their removal by polymerase β.^[21] For double-strand break (DSB) repair via homologous recombination (HR), exonucleases drive 5' end resection to produce 3' single-stranded DNA tails that invade homologous templates. Human EXO1 performs extensive 5'→3' resection, accumulating rapidly at DSBs to recruit RPA and Rad51; its activity is regulated by ATM-dependent phosphorylation at S714, and EXO1 depletion impairs HR efficiency, increases chromosomal instability, and sensitizes cells to ionizing radiation.^[22] In yeast, bidirectional resection involves the 3'→5' exonuclease Mre11, which initiates short-range processing up to 300 nucleotides from the break, followed by long-range 5'→3' resection by EXO1, ensuring efficient HR even at blocked ends like those generated by Spo11.^[23] Exonucleases also participate in non-homologous end joining (NHEJ), a major DSB repair pathway, where they process incompatible DNA ends for ligation. The Artemis protein (DCLRE1C) exhibits intrinsic 5'→3' exonuclease activity on single-stranded DNA and, when activated by DNA-PKcs phosphorylation, gains endonuclease function to resolve hairpins and overhangs, facilitating precise joining of broken ends during V(D)J recombination and general NHEJ. Defects in Artemis lead to radiosensitive severe combined immunodeficiency due to impaired end processing.^[24]

Exonucleases in Prokaryotes

Types in E. coli

In Escherichia coli, several key exonucleases play essential roles in DNA and RNA metabolism, with distinct directionalities and substrate preferences that contribute to processes like repair and recombination. These enzymes exemplify the diversity of prokaryotic exonucleases, often acting in a processive or distributive manner on single- or double-stranded nucleic acids.^[25] Exonuclease I (ExoI), encoded by the sbcB (or xonA) gene, is a 3′→5′ exonuclease specific for single-stranded DNA (ssDNA). It operates in a highly processive manner, capable of degrading up to thousands of nucleotides per binding event, particularly on homopolymeric substrates, and its activity is stimulated by single-stranded DNA-binding protein (SSB). ExoI contributes to DNA recombination, repair of damage, mutation avoidance, and mismatch repair by resecting ssDNA tails.^[25]^[26] Exonuclease III (ExoIII), encoded by xthA, functions as a 3′→5′ exonuclease on double-stranded DNA (dsDNA), exhibiting a distributive mechanism where it releases mononucleotides stepwise but frequently dissociates from the substrate. Beyond exonuclease activity, ExoIII possesses 3′ phosphomonoesterase function to remove phosphate groups from 3′ termini and apurinic/apyrimidinic (AP) endonuclease activity to cleave at abasic sites, aiding in the removal of damaged residues. It is primarily involved in base excision repair and the response to oxidative or ionizing radiation-induced DNA damage.^[25]^[27] RecJ is a 5′→3′ exonuclease with strict specificity for ssDNA, degrading substrates in a processive fashion at rates of several thousand nucleotides per minute, and its activity is enhanced by SSB. Unlike many other exonucleases, RecJ requires Mg²⁺ for function and plays a critical role in homologous recombination by resecting 5′ ends to generate 3′ ssDNA tails for RecA-mediated strand invasion, as well as in DNA repair and mismatch repair pathways.^[25]^[28] Polynucleotide phosphorylase (PNPase), encoded by pnp, is a 3′→5′ exoribonuclease that targets single-stranded RNA rather than DNA, employing a phosphorolytic mechanism that uses inorganic phosphate to cleave phosphodiester bonds and release nucleoside diphosphates. This enzyme forms a homotrimeric complex and is integral to mRNA turnover and quality control in E. coli, often working in concert with endonucleases like RNase E to degrade transcripts processively from the 3′ end.^[29]

dnaQ Gene and Epsilon Subunit

The dnaQ gene, also known as mutD, encodes the ε (epsilon) subunit of the DNA polymerase III (Pol III) holoenzyme in Escherichia coli. This subunit is one of the three core components of Pol III, alongside the α (polymerase) and θ subunits, and plays a critical role in the replicative DNA synthesis machinery of the bacterium.^[30]^[31] The dnaQ gene was first identified in the late 1970s through studies of conditional lethal mutator mutants. In 1974, the mutD locus was described as a conditional mutator gene that dramatically elevates spontaneous mutation rates when active, mapped to approximately 5.7 minutes on the E. coli chromosome. Subsequent work in 1978 isolated the dnaQ49 allele, a temperature-sensitive mutation conferring a strong mutator phenotype at non-permissive temperatures, confirming its location near mutD and linking it to defects in DNA replication fidelity.^[32] By 1983, genetic and biochemical analyses established that dnaQ and mutD are alleles of the same gene, with the ε subunit product exhibiting 3′→5′ exonuclease activity essential for proofreading during replication.^[33]^[31] The primary function of the ε subunit is to provide 3′→5′ exonuclease activity, which removes mismatched nucleotides incorporated by the α subunit during DNA synthesis, thereby enhancing replication fidelity. This proofreading mechanism hydrolyzes the phosphodiester bond at the 3′ end of the growing DNA strand, excising errors to prevent their fixation as mutations.^[30]^[31] Mutations in dnaQ, such as mutD5 and dnaQ49, impair this exonuclease activity, resulting in a mutator phenotype that increases the spontaneous mutation rate by 10- to 1,000-fold, depending on the allele and growth conditions.^[32]^[34] These defects lead to elevated error rates across all base substitutions and frameshifts, underscoring the ε subunit's central role in maintaining genomic stability during E. coli replication.^[33]

Exonucleases in Eukaryotes

Discoveries in Humans

The identification of TREX1, a 3'-5' DNA exonuclease, marked a significant advance in understanding innate immune regulation and autoimmune disorders in humans. In 2006, researchers discovered that biallelic mutations in the TREX1 gene cause Aicardi-Goutières syndrome (AGS), a hereditary encephalopathy mimicking viral infection due to impaired degradation of cytosolic DNA, leading to excessive type I interferon production.^[35] TREX1 functions primarily in the cytoplasm to excise nucleotides from double-stranded DNA ends, preventing accumulation of self-DNA that activates nucleic acid sensors.^[35] Human exonuclease 1 (EXO1), characterized as a 5'-3' exonuclease, was cloned in 1998 and shown to interact with mismatch repair (MMR) proteins like MSH2, establishing its role in post-replicative DNA repair.^[36] In the 1990s, MRE11 was isolated as the human homolog of yeast Mre11, revealing its 3'-5' exonuclease activity within the MRN complex (MRE11-RAD50-NBS1) essential for double-strand break (DSB) repair.^[37] The complex, identified through biochemical associations in 1996, processes DSB ends for homologous recombination and non-homologous end joining, with MRE11's nuclease domain initiating resection.^[38] Mutations in MRE11, reported in the late 1990s, are associated with ataxia-telangiectasia-like disorder, underscoring its role in maintaining genomic stability and preventing neurodegeneration.^[37] Among RNA exonucleases, XRN1, the human 5'-3' exoribonuclease cloned in the mid-1990s as a yeast Xrn1 homolog, plays a central role in cytoplasmic mRNA decay by degrading decapped transcripts following deadenylation. Its activity ensures rapid turnover of aberrant or regulatory mRNAs, influencing gene expression and cellular responses to stress, with disruptions linked to altered mRNA stability in human diseases including cancer.^[39]

Discoveries in Yeast

In Saccharomyces cerevisiae, genetic screens for mutants defective in DNA repair, recombination, and replication have revealed key exonucleases central to eukaryotic genome maintenance. These studies, leveraging yeast's tractable genetics, highlighted enzymes involved in processing DNA ends and RNA transcripts, providing mechanistic insights applicable to higher eukaryotes. The Exo1 exonuclease was isolated in 1997 through a screen for genes interacting with the mismatch repair protein Msh2. This 5'-3' exonuclease plays a critical role in resecting double-strand breaks (DSBs) from the 5' end, facilitating homologous recombination during meiosis and mitotic DNA repair. Exo1-dependent resection generates single-stranded DNA tails essential for Rad51 filament formation and strand invasion, with mutants showing reduced crossover formation and increased sensitivity to DNA-damaging agents. Yeast Exo1 shares homology with human EXO1, underscoring conserved functions in resection pathways. The Mre11-Rad50-Xrs2 (MRX) complex emerged from radiation-sensitive and meiotic recombination-defective mutants identified in the 1980s and 1990s. RAD50 was first characterized from early screens for DSB repair defects, encoding an ATP-dependent DNA-binding protein that bridges DNA ends. Mre11, cloned in the early 1990s, contributes 3'-5' exonuclease and endonuclease activities, initiating DSB processing by nicking 5' strands near breaks. Xrs2, identified in 1992, recruits the complex to chromatin and mediates checkpoint signaling. Together, MRX performs initial endonucleolytic cleavage followed by limited exonucleolytic resection, promoting extensive 5'-3' processing by downstream nucleases like Exo1; mutants exhibit defective DSB repair, telomere maintenance, and meiotic progression. Rat1, a nuclear 5'-3' exoribonuclease homologous to cytoplasmic Xrn1, was linked to transcription termination in the 2000s via studies on RNA polymerase II (Pol II) release. Genetic and biochemical analyses demonstrated that Rat1 degrades nascent RNA downstream of the polyadenylation site, propelling the "torpedo" mechanism to displace Pol II from DNA. Rat1 associates with the Rai1 cofactor for efficient activity, and its depletion causes Pol II accumulation at gene ends, disrupting termination and rRNA processing; this function is distinct from Xrn1's primary role in bulk mRNA decay. Dna2, a bifunctional helicase-nuclease, was discovered in 1995 from temperature-sensitive replication mutants, with its nuclease activity confirmed shortly thereafter. This enzyme unwinds DNA via 5'-3' helicase activity while exhibiting endonuclease and 3'-5' exonuclease functions, primarily processing RNA/DNA flaps at replication forks during Okazaki fragment maturation. Dna2 collaborates with the flap endonuclease FEN1 to remove primer RNA, preventing fork stalling; mutants accumulate replication intermediates and show synthetic lethality with other processing factors.

Exonucleases in Viruses

Role in Coronaviruses

In coronaviruses, the nonstructural protein 14 (nsp14), also known as ExoN, functions as a 3'–5' exoribonuclease that plays a pivotal role in maintaining the fidelity of viral RNA replication. This enzyme removes mismatched nucleotides incorporated during RNA synthesis by the viral RNA-dependent RNA polymerase, thereby acting as a proofreading mechanism that is rare among RNA viruses.^[40] The ExoN activity is enhanced when nsp14 forms a complex with nsp10, which stimulates its exonuclease function by over 35-fold, allowing for efficient error correction and contributing to the low mutation rate observed in coronaviruses despite their large genomes of approximately 30 kb.^[41] This proofreading capability, first characterized in studies of SARS-CoV in the early 2000s, enables genome stability and supports the evolution of diverse coronavirus species without the high error rates typical of other RNA viruses.^[42] The ExoN domain of nsp14 is structurally conserved across coronaviruses, including SARS-CoV, MERS-CoV, and SARS-CoV-2, and its catalytic activity is essential for efficient viral replication. Mutations that abolish ExoN function lead to increased mutation rates and attenuated viral growth, as demonstrated in cell culture and animal models for both SARS-CoV and MERS-CoV.^[43] Structural analyses reveal that ExoN recognizes mismatched base pairs through specific interactions in its active site, preferentially excising them from the 3' end of nascent RNA strands.^[44] This high-fidelity replication is particularly advantageous for coronaviruses, allowing them to maintain genetic integrity over their expansive genomes and adapt to host pressures without excessive variability.^[45] While nsp14 provides proofreading, nsp15 (EndoU) serves a complementary role as a uridine-specific endoribonuclease that primarily cleaves viral RNA to evade host innate immune detection. Nsp15 processes double-stranded RNA intermediates produced during replication, reducing the accumulation of pathogen-associated molecular patterns that would otherwise trigger interferon responses.^[46] Its endonucleolytic activity is conserved across coronaviruses and is crucial for virulence, as evidenced by studies showing that nsp15 mutants elicit stronger host antiviral defenses and exhibit reduced pathogenicity in vivo.^[47] Together, these nucleases ensure robust viral propagation by balancing replication accuracy with immune suppression.

Mechanisms in Other Viruses

In bacteriophage λ, the lambda exonuclease (λ Exo) plays a crucial role in the viral recombination system known as Red. This enzyme functions as a highly processive 5'→3' exonuclease that binds to the ends of double-stranded DNA (dsDNA) and selectively degrades the 5'-phosphorylated strand, generating long 3' single-stranded DNA (ssDNA) overhangs essential for homologous recombination.^[48] The processivity of λ Exo arises from its ring-shaped homotrimeric structure, which allows it to thread along the DNA substrate while hydrolyzing phosphodiester bonds without dissociation, enabling the degradation of thousands of nucleotides per binding event.^[49] These ssDNA tails facilitate annealing with homologous sequences, promoting efficient recombination during the phage lytic cycle.^[50] Picornaviruses, such as poliovirus, exhibit strategies to evade the host's 5'→3' RNA exoribonuclease Xrn1, which targets uncapped viral RNAs for decay. During infection, Xrn1 undergoes proteasomal degradation, thereby preventing the enzyme from digesting unlinked viral positive-sense RNA genomes that lack a 5' cap.^[51] Additionally, the viral protein VPg covalently linked to the 5' end of picornavirus RNA resists decapping by host Dcp2, further reducing susceptibility to Xrn1-mediated degradation and stabilizing the genome for replication.^[51] These mechanisms allow picornaviruses to counteract the antiviral effects of cytoplasmic RNA decay pathways, contrasting with coronaviral proofreading exonucleases that enhance RNA fidelity.^[51] In HIV-1, integration of the viral DNA into the host genome involves association with the host 3'→5' exonuclease TREX1, which degrades unprocessed blunt-ended viral DNA generated during reverse transcription. TREX1 preferentially degrades these non-integratable forms while sparing 3'-processed ends created by the viral integrase, thereby promoting efficient strand transfer and reducing autointegration.^[52] This exonuclease activity reduces autointegration of viral DNA and enhances productive infection, with TREX1 levels upregulated in infected cells to support the process.^[52] The hepatitis B virus (HBV) polymerase incorporates an intrinsic 3'→5' exonuclease activity within its RNase H domain, critical for reverse transcription of the pregenomic RNA. This activity degrades the RNA strand in the RNA-DNA hybrid following initial endonucleolytic cleavage, exposing the template for plus-strand DNA synthesis and ensuring complete genome replication.^[53] The exonuclease function is tightly coupled to the polymerase domain, exhibiting specificity for RNA in hybrids and contributing to the virus's unique replication strategy in hepatocytes.^[53]

Historical and Recent Developments

Early Discoveries

The initial characterizations of exonucleases emerged in the mid-20th century, with early studies focusing on enzymes from eukaryotic sources such as snake venom. In the 1950s and 1960s, researchers identified phosphodiesterases from rattlesnake (Crotalus adamanteus) and other snake venoms that exhibited 3'→5' exonuclease activity, hydrolyzing oligonucleotides from the 3'-hydroxyl terminus to produce 5'-mononucleotides.^[54] These venom-derived enzymes, purified and characterized for their substrate specificity on both ribo- and deoxyribo-oligonucleotides, represented the first documented eukaryotic exonucleases and served as tools for nucleic acid sequencing and structural analysis. In prokaryotes, significant progress occurred in the 1960s with the discovery of Exonuclease III (Exo III) in Escherichia coli. Weiss and Richardson identified this enzyme in 1967 as part of a repair system in T4 bacteriophage-infected E. coli extracts, where it demonstrated 3'→5' exonuclease activity on double-stranded DNA, progressively removing nucleotides from 3'-hydroxyl ends while also possessing AP endonuclease and 3'-phosphatase functions essential for DNA repair.^[55] This bifunctional activity was crucial for processing single-strand breaks in transforming DNA, marking a key step in understanding bacterial DNA metabolism. The 1970s brought further insights into exonuclease activities associated with DNA polymerases in E. coli. Brutlag and Kornberg characterized the intrinsic 3'→5' exonuclease activity of DNA Polymerase I (Pol I) in 1972, showing its role in proofreading by selectively hydrolyzing mismatched nucleotides at the 3'-end of growing DNA chains, thereby enhancing replication fidelity.^[56] This polymerase-associated exonuclease, previously noted in purifications but fully delineated here, complemented earlier work on Pol I's 5'→3' exonuclease domain. Early investigations into viral exonucleases also began in the 1960s, with key contributions from the Kornberg laboratory on bacteriophage T4. Gomatos and Kornberg described exonuclease activities in T4-infected E. coli in the early 1960s, linking them to viral DNA replication and maturation processes, which laid groundwork for understanding phage-specific nucleases.

Advances Since 2000

Since 2000, advances in exonuclease research have been driven by high-resolution structural studies, integration into genome-editing technologies, and exploration of therapeutic targets, particularly in viral infections and cancer. A pivotal development was the determination of the crystal structure of human TREX1, a 3'–5' DNA exonuclease implicated in autoimmune diseases, at 2.0 Å resolution in 2007. This structure revealed the enzyme's active site architecture, including two metal ions coordinating the catalytic residues and a polyproline II helix for protein-protein interactions, explaining its substrate specificity and 3' nucleotide bias.^[57] Subsequent structures, such as those of TREX1 in complex with DNA substrates, further elucidated overhang excision mechanisms and disease-related mutations, enabling targeted inhibitor design. In the 2010s, exonucleases emerged as key modulators in CRISPR-Cas systems, enhancing editing precision and efficiency by processing double-strand breaks (DSBs) generated by Cas nucleases. For instance, fusion of 5'–3' exonucleases like RecJ to Cas9 variants increased homology-directed repair (HDR) efficiency (e.g., up to 1.8-fold in some loci) while altering indel formation, as demonstrated in human cell lines.^[58] These engineered exonuclease editors promoted precise insertions and deletions by resecting DSB ends, minimizing off-target effects and boosting therapeutic potential in gene correction, with applications reported in models of genetic disorders. The COVID-19 pandemic accelerated structural insights into viral exonucleases, notably SARS-CoV-2 nsp14, a bifunctional 3'–5' exonuclease-methyltransferase essential for viral replication fidelity. Crystal structures of the nsp14-nsp10 complex, resolved at 1.6 Å in 2021, highlighted the exonuclease domain's RNA-binding cleft and catalytic triad, showing how nsp10 allosterically activates proofreading to excise mismatched nucleotides.^[45] Follow-up studies from 2020–2023, including a 2.5 Å structure with mismatched RNA, identified features relevant to antiviral design, paving the way for compounds that disrupt exonuclease activity and increase mutation rates in coronaviruses.^[44] In the 2020s, exonucleases like EXO1 have gained prominence in cancer research, where pan-cancer analyses confirmed EXO1 upregulation correlates with poor prognosis and immunosuppressive microenvironments across multiple cancers, positioning it as a potential biomarker for immunotherapy response.^[59]

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Crystal structures of Escherichia coli exonuclease I in complex with ...
Apr 22, 2013 · Escherichia coli Exonuclease I (ExoI) digests single-stranded DNA (ssDNA) in the 3′-5′ direction in a highly processive manner.Missing: directionality | Show results with:directionality
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Proofreading exonuclease activity of human DNA polymerase δ and ...
Replicative DNA polymerases possess 3′ → 5′ exonuclease activity to reduce misincorporation of incorrect nucleotides by proofreading during replication.
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Switching between polymerase and exonuclease sites in DNA ... - NIH
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Structure and function relationships in mammalian DNA polymerases
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The proofreading 3'-->5' exonuclease activity of DNA polymerases
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A conserved 5' to 3' exonuclease activity in the yeast and ... - PubMed
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Human exonuclease 1 connects nucleotide excision repair (NER ...
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5'-exonuclease activity of human apurinic endonuclease (Ape1) by ...
The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA.
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We show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and ...
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Bidirectional resection of DNA double-strand breaks by Mre11 and ...
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RNA recognition by 3'-to-5' exonucleases: the substrate perspective
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Genome-wide analysis reveals distinct substrate specificities of Rrp6 ...
The exosome complex is also involved in the surveillance of mRNAs in both the nucleus and the cytoplasm, including nonsense-mediated decay (NMD) targets. The ...
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Identification of the epsilon-subunit of Escherichia coli DNA ... - PNAS
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A New Conditional Lethal Mutator (dnaQ49) in Escherichia Coli K12
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Mutator strains of Escherichia coli, mutD and dnaQ, with ... - PNAS
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Mutational Signatures in Wild Type Escherichia coli Strains Reveal ...
On a rich medium, mutants that carry an almost inactive DnaQ have a 10 to 1,000-fold higher mutation rate than the WT strains (Cox and Horner 1982).Missing: increase | Show results with:increase
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Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 ...
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Identification of a Human Gene Encoding a Homologue of ...
The EXO1 gene was identified in Saccharomyces cerevisiae as a gene encoding an exonuclease that interacts with MSH2 and functions in mismatch repair and ...
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Isolation and characterization of the human MRE11 homologue
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The exonuclease Xrn1 activates transcription and translation of ...
Mar 21, 2019 · The highly conserved 5'–3' exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay.
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Coronavirus RNA Proofreading: Molecular Basis and Therapeutic ...
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Coronavirus RNA Proofreading: Molecular Basis and Therapeutic ...
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Coronavirus genomic nsp14‐ExoN, structure, role, mechanism, and ...
This exonucleolytic activity is crucial for the Coronavirus replication proofreading activity, a property lacking in other RNA viruses that improve its fidelity ...
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The Enzymatic Activity of the nsp14 Exoribonuclease Is Critical for ...
Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis.
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Structural basis of mismatch recognition by a SARS-CoV-2 ... - Science
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Structure and dynamics of SARS-CoV-2 proofreading ... - PNAS
SARS-CoV-2 nonstructural protein 14 (nsp14) exoribonuclease (ExoN) plays important roles in the proofreading during viral RNA synthesis and the evasion of ...
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Coronavirus nonstructural protein 15 mediates evasion of dsRNA ...
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Cryo-EM structures of the SARS-CoV-2 endoribonuclease Nsp15 ...
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The enzymatic basis of processivity in λ exonuclease - PMC
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Structure and mechanism of the Red recombination system of ...
Overview of the Red recombination system from bacteriophage λ. λ exo forms a ring-shaped homotrimer that binds to dsDNA ends and processively digests the 5′- ...
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Single molecule analysis reveals three phases of DNA degradation ...
Here, we use single molecule FRET to reveal three phases of λ exonuclease reactions: initiation, distributive and processive phases.
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Diverse Strategies Used by Picornaviruses to Escape Host RNA ...
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Human Three Prime Repair Exonuclease 1 Promotes HIV-1 ... - NIH
Human three prime repair exonuclease 1 promotes HIV-1 integration by preferentially degrading unprocessed viral DNA.
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Purification and enzymatic characterization of the hepatitis B virus ...
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Structural basis for overhang excision and terminal unwinding of ...
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Enhanced genome editing efficiency of CRISPR PLUS - Nature
Aug 10, 2021 · Increased genome-editing efficiency of Cas9 chimeric fusion with RecJ or GFP. To prepare Cas9 chimeric fusion proteins, we translationally ...
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Exonuclease editor promotes precision of gene editing in ... - NIH
May 20, 2024 · In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of ...
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EXO1's pan-cancer roles: diagnostic, prognostic, and immunological ...
Mar 12, 2025 · EXO1 was found to be upregulated in multiple cancers, with significant diagnostic potential as indicated by high AUC values in ROC analyses.