Knowing when to stop: Transcription termination on protein-coding genes by eukaryotic RNAPII.

Gene expression is controlled in a dynamic and regulated manner to allow for the consistent and steady expression of some proteins as well as the rapidly changing production of other proteins. Transcription initiation has been a major focus of study because it is highly regulated. However, termination of transcription also plays an important role in controlling gene expression. Transcription termination on protein-coding genes is intimately linked with 3' end cleavage and polyadenylation of transcripts, and it generally results in the production of a mature mRNA that is exported from the nucleus. Termination on many non-coding genes can also result in the production of a mature transcript. Termination is dynamically regulated-premature termination and transcription readthrough occur in response to a number of cellular signals, and these can have varied consequences on gene expression. Here, we review eukaryotic transcription termination by RNA polymerase II (RNAPII), focusing on protein-coding genes.

Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.

DNA polymerase ε (Polε) carries out high-fidelity leading strand synthesis owing to its exonuclease activity. Polε polymerase and exonuclease activities are balanced, because of partitioning of nascent DNA strands between catalytic sites, so that net resection occurs when synthesis is impaired. In vivo, DNA synthesis stalling activates replication checkpoint kinases, which act to preserve the functional integrity of replication forks. We show that stalled Polε drives nascent strand resection causing fork functional collapse, averted via checkpoint-dependent phosphorylation. Polε catalytic subunit Pol2 is phosphorylated on serine 430, influencing partitioning between polymerase and exonuclease active sites. A phosphormimetic S430D change reduces exonucleolysis in vitro and counteracts fork collapse. Conversely, non-phosphorylatable pol2-S430A expression causes resection-driven stressed fork defects. Our findings reveal that checkpoint kinases switch Polε to an exonuclease-safe mode preventing nascent strand resection and stabilizing stalled replication forks. Elective partitioning suppression has implications for the diverse Polε roles in genome integrity maintenance.

EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart.

Stalled DNA replication fork restart after stress as orchestrated by ATR kinase, BLM helicase, and structure-specific nucleases enables replication, cell survival, and genome stability. Here we unveil human exonuclease V (EXO5) as an ATR-regulated DNA structure-specific nuclease and BLM partner for replication fork restart. We find that elevated EXO5 in tumors correlates with increased mutation loads and poor patient survival, suggesting that EXO5 upregulation has oncogenic potential. Structural, mechanistic, and mutational analyses of EXO5 and EXO5-DNA complexes reveal a single-stranded DNA binding channel with an adjacent ATR phosphorylation motif (T88Q89) that regulates EXO5 nuclease activity and BLM binding identified by mass spectrometric analysis. EXO5 phospho-mimetic mutant rescues the restart defect from EXO5 depletion that decreases fork progression, DNA damage repair, and cell survival. EXO5 depletion furthermore rescues survival of FANCA-deficient cells and indicates EXO5 functions epistatically with SMARCAL1 and BLM. Thus, an EXO5 axis connects ATR and BLM in directing replication fork restart.

Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting.

The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.

Discrete RNA-DNA hybrid cleavage by the EXD2 exonuclease pinpoints two rate-limiting steps.

EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double-stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding-degradation coordination of an exonuclease. Here, using a single-molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA-DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2-mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4-bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate-limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases.

Structure of Escherichia coli exonuclease VII.

Exonuclease VII (ExoVII) is a ubiquitous bacterial nuclease. Encoded by the xseA and xseB genes, ExoVII participates in multiple nucleic acid-dependent pathways including the processing of multicopy single-stranded DNA and the repair of covalent DNA-protein crosslinks (DPCs). Although many biochemical properties of ExoVII have been defined, little is known about its structure/function relationships. Here, we use cryoelectron microscopy (cryoEM) to determine that Escherichia coli ExoVII comprises a highly elongated XseA4·XseB24 holo-complex. Each XseA subunit dimerizes through a central extended α-helical segment decorated by six XseB subunits and a C-terminal, domain-swapped ß-barrel element; two XseA2·XseB12 subcomplexes further associate using N-terminal OB (oligonucleotide/oligosaccharide-binding) folds and catalytic domains to form a spindle-shaped, catenated octaicosamer. The catalytic domains of XseA, which adopt a nuclease fold related to 3-dehydroquinate dehydratases, are sequestered in the center of the complex and accessible only through large pores formed between XseA tetramers. The architectural organization of ExoVII, combined with biochemical studies, indicate that substrate selectivity is controlled by steric access to its nuclease elements and that tetramer dissociation results from substrate DNA binding. Despite a lack of sequence and fold homology, the physical organization of ExoVII is reminiscent of Mre11·Rad50/SbcCD ATP (adenosine triphosphate)-dependent nucleases used in the repair of double-stranded DNA breaks, including those formed by DPCs through aberrant topoisomerase activity, suggesting that there may have been convergent evolutionary pressure to contend with such damage events.

Independent neofunctionalization of Dxo1 in Saccharomyces and Candida led to 25S rRNA processing function.

Eukaryotic genomes typically encode one member of the DXO/Dxo1/Rai1 family of enzymes, which can hydrolyze the 5' ends of RNAs with a variety of structures that deviate from the canonical 7mGpppN. In contrast, the Saccharomyces genome encodes two family members and the second copy, Dxo1, is a distributive 5' exoribonuclease that is required for the final maturation of the 5' end of 25S rRNA from a 25S' precursor. Here we show that this 25S rRNA maturation function is not conserved across kingdoms, but arose in the budding yeasts. Interestingly, the origin of 25S processing capacity coincides with the duplication of this gene and this capacity is absent in the nonduplicated genes. Strikingly, two different clades of budding yeasts have undergone parallel evolution: Both duplicated their DXO/Dxo1/Rai1 gene, and in both cases one copy gained the 25S processing function. This was accompanied by many parallel sequence changes, a remarkable case of reproducible neofunctionalization.

mRNA Deadenylation Is Coupled to Translation Rates by the Differential Activities of Ccr4-Not Nucleases.

Translation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases-Ccr4 and Caf1/Pop2-that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes. Surprisingly, Pab1 release relies on Ccr4 activity. In agreement with this, in vivo experiments in budding yeast show that Ccr4 is a general deadenylase that acts on all mRNAs. In contrast, Caf1 only trims poly(A) not bound by Pab1. As a consequence, Caf1 is a specialized deadenylase required for the selective deadenylation of transcripts with lower rates of translation elongation and reduced Pab1 occupancy. These findings reveal a coupling between the rates of translation and deadenylation that is dependent on Pab1 and Ccr4-Not.

The enzymes for genome size increase and maintenance of large (+)RNA viruses.

With sizes <50 kb, viral RNA genomes are at the crossroads of genetic, biophysical, and biochemical stability in their host cell. Here, we analyze the enzymatic assets accompanying large RNA genome viruses, mostly based on recent scientific advances in Coronaviridae. We argue that, in addition to the presence of an RNA exonuclease (ExoN), two markers for the large size of viral RNA genomes are (i) the presence of one or more RNA methyltransferases (MTases) and (ii) a specific architecture of the RNA-dependent RNA polymerase active site. We propose that RNA genome expansion and maintenance are driven by an evolutionary ménage-à-trois made of fast and processive RNA polymerases, RNA repair ExoNs, and RNA MTases that relates to the transition between RNA- to DNA-based life.

Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties.

HIV-1 integration into the human genome is dependent on 3'-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3'-processed DNA remained resistant. Here, we describe the mechanism by which the 3'-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3'-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3'-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼2-fold) for the 3'-processed DNA than the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3'-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3'-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3'-processed HIV-1 DNA.

Increasing deletion sizes and the efficiency of CRISPR/Cas9-mediated mutagenesis by SunTag-mediated TREX1 recruitment.

Previously, it has been shown that mutagenesis frequencies can be improved by directly fusing the human exonuclease TREX2 to Cas9, resulting in a strong increase in the frequency of smaller deletions at the cut site. Here, we demonstrate that, by using the SunTag system for recruitment of TREX2, the mutagenesis efficiency can be doubled in comparison to the direct fusion in Arabidopsis thaliana. Therefore, we also tested the efficiency of the system for targeted deletion formation by recruiting two other 3'-5' exonucleases, namely the human TREX1 and E. coli ExoI. It turns out that SunTag-mediated recruitment of TREX1 not only improved the general mutation induction efficiency slightly in comparison to TREX2, but that, more importantly, the mean size of the induced deletions was also enhanced, mainly via an increase of deletions of 25 bp or more. EcExoI also yielded a higher amount of larger deletions. However, only in the case of TREX1 and TREX2, the effect was predominately SunTag-dependent, indicating efficient target-specific recruitment. Using SunTag-mediated TREX1 recruitment at other genomic sites, we were able to obtain similar deletion patterns. Thus, we were able to develop an attractive novel editing tool that is especially useful for obtaining deletions in the range from 20 to 40 bp around the cut site. Such sizes are often required for the manipulation of cis-regulatory elements. This feature is closing an existing gap as previous approaches, based on single nucleases or paired nickases or nucleases, resulted in either shorter or longer deletions, respectively.

Alternative translation contributes to the generation of a cytoplasmic subpopulation of the Junín virus nucleoprotein that inhibits caspase activation and innate immunity.

The highly pathogenic arenavirus, Junín virus (JUNV), expresses three truncated alternative isoforms of its nucleoprotein (NP), i.e., NP53kD, NP47kD, and NP40kD. While both NP47kD and NP40kD have been previously shown to be products of caspase cleavage, here, we show that expression of the third isoform NP53kD is due to alternative in-frame translation from M80. Based on this information, we were able to generate recombinant JUNVs lacking each of these isoforms. Infection with these mutants revealed that, while all three isoforms contribute to the efficient control of caspase activation, NP40kD plays the predominant role. In contrast to full-length NP (i.e., NP65kD), which is localized to inclusion bodies, where viral RNA synthesis takes place, the loss of portions of the N-terminal coiled-coil region in these isoforms leads to a diffuse cytoplasmic distribution and a loss of function in viral RNA synthesis. Nonetheless, NP53kD, NP47kD, and NP40kD all retain robust interferon antagonistic and 3'-5' exonuclease activities. We suggest that the altered localization of these NP isoforms allows them to be more efficiently targeted by activated caspases for cleavage as decoy substrates, and to be better positioned to degrade viral double-stranded (ds)RNA species that accumulate in the cytoplasm during virus infection and/or interact with cytosolic RNA sensors, thereby limiting dsRNA-mediated innate immune responses. Taken together, this work provides insight into the mechanism by which JUNV leverages apoptosis during infection to generate biologically distinct pools of NP and contributes to our understanding of the expression and biological relevance of alternative protein isoforms during virus infection.IMPORTANCEA limited coding capacity means that RNA viruses need strategies to diversify their proteome. The nucleoprotein (NP) of the highly pathogenic arenavirus Junín virus (JUNV) produces three N-terminally truncated isoforms: two (NP47kD and NP40kD) are known to be produced by caspase cleavage, while, here, we show that NP53kD is produced by alternative translation initiation. Recombinant JUNVs lacking individual NP isoforms revealed that all three isoforms contribute to inhibiting caspase activation during infection, but cleavage to generate NP40kD makes the biggest contribution. Importantly, all three isoforms retain their ability to digest double-stranded (ds)RNA and inhibit interferon promoter activation but have a diffuse cytoplasmic distribution. Given the cytoplasmic localization of both aberrant viral dsRNAs, as well as dsRNA sensors and many other cellular components of innate immune activation pathways, we suggest that the generation of NP isoforms not only contributes to evasion of apoptosis but also robust control of the antiviral response.

Activation of protein kinase receptor (PKR) plays a pro-viral role in mammarenavirus-infected cells.

Many viruses, including mammarenaviruses, have evolved mechanisms to counteract different components of the host cell innate immunity, which is required to facilitate robust virus multiplication. The double-stranded RNA (dsRNA) sensor protein kinase receptor (PKR) pathway plays a critical role in the cell anti-viral response. Whether PKR can restrict the multiplication of the Old World mammarenavirus lymphocytic choriomeningitis virus (LCMV) and the mechanisms by which LCMV may counteract the anti-viral functions of PKR have not yet been investigated. Here we present evidence that LCMV infection results in very limited levels of PKR activation, but LCMV multiplication is enhanced in the absence of PKR. In contrast, infection with a recombinant LCMV with a mutation affecting the 3'-5' exonuclease (ExoN) activity of the viral nucleoprotein resulted in robust PKR activation in the absence of detectable levels of dsRNA, which was associated with severely restricted virus multiplication that was alleviated in the absence of PKR. However, pharmacological inhibition of PKR activation resulted in reduced levels of LCMV multiplication. These findings uncovered a complex role of the PKR pathway in LCMV-infected cells involving both pro- and anti-viral activities.IMPORTANCEAs with many other viruses, the prototypic Old World mammarenavirus LCMV can interfere with the host cell innate immune response to infection, which includes the dsRNA sensor PKR pathway. A detailed understanding of LCMV-PKR interactions can provide novel insights about mammarenavirus-host cell interactions and facilitate the development of effective anti-viral strategies against human pathogenic mammarenaviruses. In the present work, we present evidence that LCMV multiplication is enhanced in PKR-deficient cells, but pharmacological inhibition of PKR activation unexpectedly resulted in severely restricted propagation of LCMV. Likewise, we document a robust PKR activation in LCMV-infected cells in the absence of detectable levels of dsRNA. Our findings have revealed a complex role of the PKR pathway during LCMV infection and uncovered the activation of PKR as a druggable target for the development of anti-viral drugs against human pathogenic mammarenaviruses.

Splicing inactivation generates hybrid mRNA-snoRNA transcripts targeted by cytoplasmic RNA decay.

Many small nucleolar RNAs (snoRNA)s are processed from introns of host genes, but the importance of splicing for proper biogenesis and the fate of the snoRNAs is not well understood. Here, we show that inactivation of splicing factors or mutation of splicing signals leads to the accumulation of partially processed hybrid messenger RNA-snoRNA (hmsnoRNA) transcripts. hmsnoRNAs are processed to the mature 3' ends of the snoRNAs by the nuclear exosome and bound by small nucleolar ribonucleoproteins. hmsnoRNAs are unaffected by translation-coupled RNA quality-control pathways, but they are degraded by the major cytoplasmic exonuclease Xrn1p, due to their messenger RNA (mRNA)-like 5' extensions. These results show that completion of splicing is required to promote complete and accurate processing of intron-encoded snoRNAs and that splicing defects lead to degradation of hybrid mRNA-snoRNA species by cytoplasmic decay, underscoring the importance of splicing for the biogenesis of intron-encoded snoRNAs.

Exonuclease editor promotes precision of gene editing in mammalian cells.

BACKGROUND: Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. RESULTS: Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. CONCLUSIONS: Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

Kinetics of DNA strand transfer between polymerase and proofreading exonuclease active sites regulates error correction during high-fidelity replication.

We show that T7 DNA polymerase (pol) and exonuclease (exo) domains contribute to selective error correction during DNA replication by regulating bidirectional strand transfer between the two active sites. To explore the kinetic basis for selective removal of mismatches, we used a fluorescent cytosine analog (1,3-diaza-2-oxophenoxazine) to monitor the kinetics of DNA transfer between the exo and pol sites. We globally fit stopped-flow fluorescence and base excision kinetic data and compared results obtained with ssDNA versus duplex DNA to resolve how DNA transfer governs exo specificity. We performed parallel studies using hydrolysis-resistant phosphorothioate oligonucleotides to monitor DNA transfer to the exo site without hydrolysis. ssDNA binds to the exo site at the diffusion limit (109 M-1 s-1, Kd = 40 nM) followed by fast hydrolysis of the 3'-terminal nucleotide (>5000 s-1). Analysis using duplex DNA with a 3'-terminal mismatch or a buried mismatch exposed a unique intermediate state between pol and exo active sites and revealed that transfer via the intermediate to the exo site is stimulated by free nucleoside triphosphates. Transfer from the exo site back to the pol site after cleavage is fast and efficient. We propose a model to explain why buried mismatches are removed faster than single 3'-terminal mismatches and thereby provide an additional opportunity for error correction. Our data provide the first comprehensive model to explain how DNA transfer from pol to exo active sites and back again after base excision allow efficient selective mismatch removal during DNA replication to improve fidelity by more than 1000-fold.

Characterization of organelle DNA degradation mediated by DPD1 exonuclease in the rice genome-edited line.

Mitochondria and plastids, originated as ancestral endosymbiotic bacteria, contain their own DNA sequences. These organelle DNAs (orgDNAs) are, despite the limited genetic information they contain, an indispensable part of the genetic systems but exist as multiple copies, making up a substantial amount of total cellular DNA. Given this abundance, orgDNA is known to undergo tissue-specific degradation in plants. Previous studies have shown that the exonuclease DPD1, conserved among seed plants, degrades orgDNAs during pollen maturation and leaf senescence in Arabidopsis. However, tissue-specific orgDNA degradation was shown to differ among species. To extend our knowledge, we characterized DPD1 in rice in this study. We created a genome-edited (GE) mutant in which OsDPD1 and OsDPD1-like were inactivated. Characterization of this GE plant demonstrated that DPD1 was involved in pollen orgDNA degradation, whereas it had no significant effect on orgDNA degradation during leaf senescence. Comparison of transcriptomes from wild-type and GE plants with different phosphate supply levels indicated that orgDNA had little impact on the phosphate starvation response, but instead had a global impact in plant growth. In fact, the GE plant showed lower fitness with reduced grain filling rate and grain weight in natural light conditions. Taken together, the presented data reinforce the important physiological roles of orgDNA degradation mediated by DPD1.

Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles.

The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent DNA or spontaneous partitioning of primer-end to the exonuclease site as a "cost of proofreading". Instead, we show that replication hurdles, including secondary structures in template, slowed helicase, or uncoupled helicase-polymerase, increase DNA reannealing and polymerase backtracking, and generate frayed primer-ends that are shuttled to the exonuclease site and excised efficiently. Our studies indicate that active-site shuttling occurs at a high frequency, and we propose that it serves as a proofreading mechanism to protect primer-ends from mutagenic extensions.

Crystal structure of the 3'→5' exonuclease from Methanocaldococcus jannaschii.

RecJ exonucleases are members of the DHH phosphodiesterase family ancestors of eukaryotic Cdc45, the key component of the CMG (Cdc45-MCM-GINS) complex at the replication fork. They are involved in DNA replication and repair, RNA maturation and Okazaki fragment degradation. Bacterial RecJs resect 5'-end ssDNA. Conversely, archaeal RecJs are more versatile being able to hydrolyse in both directions and acting on ssDNA as well as on RNA. In Methanocaldococcus jannaschii two RecJs were previously characterized: RecJ1 is a 5'→3' DNA exonuclease, MjaRecJ2 works only on 3'-end DNA/RNA with a preference for RNA. Here, I present the crystal structure of MjaRecJ2, solved at a resolution of 2.8 Å, compare it with the other RecJ structures, in particular the 5'→3' TkoGAN and the bidirectional PfuRecJ, and discuss its characteristics in light of the more recent knowledge on RecJs. This work adds new structural data that might improve the knowledge of these class of proteins.

Quick and affordable DNA cloning by reconstitution of Seamless Ligation Cloning Extract using defined factors.

The cloning of DNA fragments to plasmid vectors is at the heart of molecular biology. Recent developments have led to various methods utilizing homologous recombination of homology arms. Among them, Seamless Ligation Cloning Extract (SLiCE) is an affordable alternative solution that uses simple Escherichia coli lysates. However, the underlying molecular mechanisms remain unclear and the reconstitution of the extract by defined factors has not yet been reported. We herein show that the key factor in SLiCE is Exonuclease III (ExoIII), a double-strand (ds) DNA-dependent 3'-5' exonuclease, encoded by XthA. SLiCE prepared from the xthAΔ strain is devoid of recombination activity, whereas purified ExoIII alone is sufficient to assemble two blunt-ended dsDNA fragments with homology arms. In contrast to SLiCE, ExoIII is unable to digest (or assemble) fragments with 3' protruding ends; however, the addition of single-strand DNA-targeting Exonuclease T overcomes this issue. Through the combination of commercially available enzymes under optimized conditions, we achieved the efficient, reproducible, and affordable cocktail, "XE cocktail," for seamless DNA cloning. By reducing the cost and time required for DNA cloning, researchers will devote more resources to advanced studies and the careful validation of their own findings.