An Insight into the Mechanism of DNA Cleavage by DNA Endonuclease from the Hyperthermophilic Archaeon Pyrococcus furiosus.

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N6-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme-substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature.

Engineering psychrophilic polymerase for nanopore long-read sequencing.

Unveiling the potential application of psychrophilic polymerases as candidates for polymerase-nanopore long-read sequencing presents a departure from conventional choices such as thermophilic Bacillus stearothermophilus (Bst) renowned for its limitation in temperature and mesophilic Bacillus subtilis phage (phi29) polymerases for limitations in strong exonuclease activity and weak salt tolerance. Exploiting the PB-Bst fusion DNA polymerases from Psychrobacillus (PB) and Bacillus stearothermophilus (Bst), our structural and biochemical analysis reveal a remarkable enhancement in salt tolerance and a concurrent reduction in exonuclease activity, achieved through targeted substitution of a pivotal functional domain. The sulfolobus 7-kDa protein (Sso7d) emerges as a standout fusion domain, imparting significant improvements in PB-Bst processivity. Notably, this study elucidates additional functional sites regulating exonuclease activity (Asp43 and Glu45) and processivity using artificial nucleotides (Glu266, Gln283, Leu334, Glu335, Ser426, and Asp430). By disclosing the intricate dynamics in exonuclease activity, strand displacement, and artificial nucleotide-based processivity at specific functional sites, our findings not only advance the fundamental understanding of psychrophilic polymerases but also provide novel insights into polymerase engineering.

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.

Identification of DNA variants at ultra-low variant allele frequencies via Taq polymerase cleavage of wild-specific blockers.

Detecting mutations related to tumors holds immense clinical significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a challenge for the advancement of low-copy mutant ctDNA genotyping in cancer cases. To address this, a Taqman qPCR strategy to identify rare mutations at low variant allele fractions (VAFs) has been developed. This strategy combines mutant-specific primers with wild-specific blockers. Diverging from other blocker-mediated PCRs, which rely on primer-induced strand displacement or the use of modified oligos resistant to Taq polymerase, our innovation is built upon the cleavage of specific blockers by Taq polymerase. Given its unique design, which does not hinge on strand displacement or base modification, we refer to this novel method as unmodified-blocker cleavage PCR (UBC-PCR). Multiple experiments consistently confirmed that variant distinction was improved significantly by introduction of 5' unmatched blockers into the reaction. Moreover, UBC-PCR successfully detected mutant DNA at VAFs as low as 0.01% across six different variant contexts. Multiplex UBC-PCR was also performed to identify a reference target and three mutations with a sensitivity of 0.01% VAFs in one single tube. In profiling the gene status from 12 lung cancer ctDNA samples and 22 thyroid cancer FNA DNA samples, UBC-PCR exhibited a 100% concordance rate with ddPCR and a commercial ARMS kit, respectively. Our work demonstrates that UBC-PCR can identify low-abundance variants with high sensitivity in multiplex reactions, independent of strand displacement and base modification. This strategy holds the potential to significantly impact clinical practice and precision medicine.

Engineering a DNA polymerase from Pyrobaculum calidifontis for improved activity, processivity and extension rate.

Positively charged amino acids in the DNA polymerase domain are important for interaction with DNA. Two potential residues in the palm domain of Pca-Pol, a DNA polymerase from Pyrobaculum calidifontis, were identified and mutated to arginine in order to improve the properties of this enzyme. The mutant proteins were heterologously produced in Escherichia coli. Biochemical characterization revealed that there was no significant difference in pH, metal ion, buffer preferences, 3' - 5' exonuclease activity and error rate of the wild-type and the mutant enzymes. However, the specific activity, processivity and extension rate of the mutant enzymes increased significantly. Specific activity of one of the mutants (G522R-E555R) was nearly 9-fold higher than that of the wild-type enzyme. These properties make G522R-E555R mutant enzyme a potential candidate for commercial applications.

Kinetic Features of 3'-5'-Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae.

In yeast Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5' side of an AP site, thereby forming a single-strand break containing 3'-OH and 5'-dRP ends. In addition, Apn2 has 3'-phosphodiesterase activity (removing 3'-blocking groups) and 3' → 5' exonuclease activity (both much stronger than its AP endonuclease activity). Nonetheless, the role of the 3'-5'-exonuclease activity of Apn2 remains unclear and presumably is involved in the repair of damage containing single-strand breaks. In this work, by separating reaction products in a polyacrylamide gel and by a stopped-flow assay, we performed a kinetic analysis of the interaction of Apn2 with various model DNA substrates containing a 5' overhang. The results allowed us to propose a mechanism for the cleaving off of nucleotides and to determine the rate of the catalytic stage of the process. It was found that dissociation of a reaction product from the enzyme active site is not a rate-limiting step in the enzymatic reaction. We determined an influence of the nature of the 3'-terminal nucleotide that can be cleaved off on the course of the enzymatic reaction. Finally, it was found that the efficiency of the enzymatic reaction is context-specific.

Quantitative methods to study helicase, DNA polymerase, and exonuclease coupling during DNA replication.

Genome replication is accomplished by highly regulated activities of enzymes in a multi-protein complex called the replisome. Two major enzymes, DNA polymerase and helicase, catalyze continuous DNA synthesis on the leading strand of the parental DNA duplex while the lagging strand is synthesized discontinuously. The helicase and DNA polymerase on their own are catalytically inefficient and weak motors for unwinding/replicating double-stranded DNA. However, when a helicase and DNA polymerase are functionally and physically coupled, they catalyze fast and highly processive leading strand DNA synthesis. DNA polymerase has a 3'-5' exonuclease activity, which removes nucleotides misincorporated in the nascent DNA. DNA synthesis kinetics, processivity, and accuracy are governed by the interplay of the helicase, DNA polymerase, and exonuclease activities within the replisome. This chapter describes quantitative biochemical and biophysical methods to study the coupling of these three critical activities during DNA replication. The methods include real-time quantitation of kinetics of DNA unwinding-synthesis by a coupled helicase-DNA polymerase complex, a 2-aminopurine fluorescence-based assay to map the precise positions of helicase and DNA polymerase with respect to the replication fork junction, and a radiometric assay to study the coupling of DNA polymerase, exonuclease, and helicase activities during processive leading strand DNA synthesis. These methods are presented here with bacteriophage T7 replication proteins as an example but can be applied to other systems with appropriate modifications.

Molecular Characterization of a DNA Polymerase from Thermus thermophilus MAT72 Phage vB_Tt72: A Novel Type-A Family Enzyme with Strong Proofreading Activity.

We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3'-5' exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysis proved that highly-conserved residues D15, E17, D78, D180, and D184 in 3'-5' exonuclease and D384 and D615 in the nucleotidyltransferase domain are critical for the enzyme's activity. Despite the source of origin, the Tt72 DNA polymerase has not proven to be highly thermoresistant, with a temperature optimum at 55 °C. Above 60 °C, the rapid loss of function follows with no activity > 75 °C. However, during heat treatment (10 min at 75 °C), trehalose, trimethylamine N-oxide, and betaine protected the enzyme against thermal inactivation. A midpoint of thermal denaturation at Tm = 74.6 °C (ΔHcal = 2.05 × 104 cal mol−1) and circular dichroism spectra > 60 °C indicate the enzyme's moderate thermal stability.

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes.

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5' to an AP-site; can recognize and process some damaged nucleosides; and possess 3'-phosphodiesterase, 3'-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions' concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3'-5' exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes.

Coexistence of endonuclease and exonuclease activities in a novel RecJ from Bacillus cereus.

BACKGROUND: All RecJ proteins are known to date only perform exonuclease activity. The present study reports that a novel RecJ protein obtained from Bacillus cereus isolated from marine sediments has both endonuclease and exonuclease activities. METHODS: Analysis of the BcRecJ expression induction in E. coli BL21 revealed that the BcRecJ protein cleaved plasmids and genomic DNA in the host cell, and led to cell death and decreased the DNA content. Further, the BcRecJ protein had the ability to degrade supercoiled plasmid DNA into circular or linear forms in vitro. Meanwhile, the BcRecJ protein loaded with an S-modified guide facilitated plasmid linearization and reduced smear formation. RESULTS: The results suggested that this novel BcRecJ protein was different from any reported RecJs and had a longer C-terminus. Testing the BcRecJ mutants indicated that the endonuclease activity was affected by two residues of BcRecJ (D561, E637) after testing the BcRecJ mutants. CONCLUSION: The discovery of the type of protein is a new breakthrough for the RecJ proteins, which has both endonuclease and exonuclease activities.

Conventional PCR assisted single-component assembly of spherical nucleic acids for simple colorimetric detection of SARS-CoV-2.

Continuous identification of suspected infectious cases is crucial to control the recent pandemic caused by the novel human coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Real-time polymerase chain reaction (real-time PCR) technology cannot be implemented easily and in large scale in some communities due to lack of resources and infrastructures. Here, we report a simple colorimetric strategy derived from linker-based single-component assembly of gold nanoparticle-core spherical nucleic acids (AuNP-core SNAs) for visual detection of PCR products of SARS-CoV-2 ribonucleic acid (RNA) template. A palindromic linker is designed based on SARS-CoV-2 specific E gene to program the identical colloidal SNAs into large assemblies along with a distinct red-to-purple color change. The linker acts as a probe of SARS-CoV-2 RNA in conventional PCR reaction. In the presence of the correct template the palindromic linker, which is complementary to a short region within the target amplicon, is cleaved by 5'-exonuclease activity of deoxyribonucleic acid (DNA) polymerase. Cleavage of the palindromic linker during the amplification process inhibits the single-component assembly formation of SNAs. So, positive and negative viral samples produce simply red and purple colors in the post PCR colorimetric test, respectively. Evaluation of the samples obtained from cases with laboratory-confirmed SARS-CoV-2 infection revealed that our assay can rival with real-time PCR method in sensitivity.

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.

Autophagy determines mtDNA copy number dynamics during starvation.

Derived from bacterial ancestors, mitochondria have maintained their own albeit strongly reduced genome, mitochondrial DNA (mtDNA), which encodes for a small and highly specialized set of genes. MtDNA exists in tens to thousands of copies packaged in numerous nucleoprotein complexes, termed nucleoids, distributed throughout the dynamic mitochondrial network. Our understanding of the mechanisms of how cells regulate the copy number of mitochondrial genomes has been limited. Here, we summarize and discuss our recent findings that Mip1/POLG (mitochondrial DNA polymerase gamma) critically controls mtDNA copy number by operating in 2 opposing modes, synthesis and, unexpectedly, degradation of mtDNA, when yeast cells face nutrient starvation. The balance of the 2 modes of Mip1/POLG and thus mtDNA copy number dynamics depends on the integrity of macroautophagy/autophagy, which sustains continuous synthesis and maintenance of mtDNA. In autophagy-deficient cells, a combination of nucleotide insufficiency and elevated mitochondrial ROS production impairs mtDNA synthesis and drives mtDNA degradation by the 3'-5'-exonuclease activity of Mip1/POLG resulting in mitochondrial genome depletion and irreversible respiratory deficiency. Abbrivations: mtDNA: mitochondrial DNA; mtDCN: mitochondrial DNA copy number.

Kinetic Features of 3'-5' Exonuclease Activity of Human AP-Endonuclease APE1.

Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5' to the damage. In addition to the AP-endonuclease activity, APE1 possesses 3'-5' exonuclease activity, which presumably is responsible for cleaning up nonconventional 3' ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3'-end nucleotide removal in the 3'-5' exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5' dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3' end of the short oligonucleotide. The impact of the 3'-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3'-5' exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3' nucleotide removal by APE1 in the 3'-5' exonuclease process is the release of the detached nucleotide from the enzyme's active site.

FAN1 protects against repeat expansions in a Fragile X mouse model.

The Fragile X-related disorders (FXDs) are members of a large group of human neurological or neurodevelopmental conditions known as the Repeat Expansion Diseases. The mutation responsible for all of these diseases is an expansion in the size of a disease-specific tandem repeat tract. However, the underlying cause of this unusual mutation is unknown. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the vicinity of the FAN1 (MIM* 613534) gene that are associated with variations in the age at onset of a number of Repeat Expansion Diseases. FAN1 is a nuclease that has both 5'-3' exonuclease and 5' flap endonuclease activities. Here we show in a model for the FXDs that Fan1-/- mice have expansions that, in some tissues including brain, are 2-3 times as extensive as they are in Fan1+/+ mice. However, no effect of the loss of FAN1 was apparent for germ line expansions. Thus, FAN1 plays an important role in protecting against somatic expansions but is either not involved in protecting against intergenerational repeat expansions or is redundant with other related enzymes. However, since loss of FAN1 results in increased expansions in brain and other somatic tissue, FAN1 polymorphisms may be important disease modifiers in those Repeat Expansion Diseases in which somatic expansion contributes to age at onset or disease severity.

CiAPEX2 and CiP0, candidates of AP endonucleases in Ciona intestinalis, have 3'-5' exonuclease activity and contribute to protection against oxidative stress.

Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions. AP sites inhibit transcription and DNA replication, and induce cell death. AP endonucleases are key enzymes in AP site repair. Several types of AP endonucleases have been reported, such as AP endonuclease 2 (APEX2) and ribosomal protein P0 (P0). However, it is not known how the functions and roles differ among AP endonucleases. To clarify the difference of roles among AP endonucleases, we conducted biochemical analysis focused on APEX2 and P0 homologues in Ciona intestinalis. Amino acid sequence analysis suggested that CiAPEX2 and CiP0 are AP endonuclease homologues. Although we could not detect AP endonuclease or 3'-phosphodiesterase activity, these two purified proteins exhibited 3'-5' exonuclease activity. This 3'-5' exonuclease activity was sensitive to ethylenediaminetetraacetic acid (EDTA), and the efficiency of this activity was influenced by the 3'-terminus of substrate DNA. Both CiAPEX2 and CiP0 degraded not only a 5'-protruding DNA end, but also nicked DNA, which is generated through AP endonuclease 1 (APEX1) cleavage. These two genes partially complemented the growth rate of AP endonuclease-deficient Escherichia coli treated with hydrogen peroxide. These results indicate that 3'-5' exonuclease activity is an evolutionarily conserved enzymatic activity of APEX2 and P0 homologues and this enzymatic activity may be important for AP endonucleases.

Germline PMS2 and somatic POLE exonuclease mutations cause hypermutability of the leading DNA strand in biallelic mismatch repair deficiency syndrome brain tumours.

Biallelic mismatch repair deficiency (bMMRD) in tumours is frequently associated with somatic mutations in the exonuclease domains of DNA polymerases POLE or POLD1, and results in a characteristic mutational profile. In this article, we describe the genetic basis of ultramutated high-grade brain tumours in the context of bMMRD. We performed exome sequencing of two second-cousin patients from a large consanguineous family of Indian origin with early onset of high-grade glioblastoma and astrocytoma. We identified a germline homozygous nonsense variant, p.R802*, in the PMS2 gene. Additionally, by genome sequencing of these tumours, we found extremely high somatic mutation rates (237/Mb and 123/Mb), as well as somatic mutations in the proofreading domain of POLE polymerase (p.P436H and p.L424V), which replicates the leading DNA strand. Most interestingly, we found, in both cancers, that the vast majority of mutations were consistent with the signature of POLE exo- , i.e. an abundance of C>A and C>T mutations, particularly in special contexts, on the leading strand. We showed that the fraction of mutations under positive selection among mutations in tumour suppressor genes is more than two-fold lower in ultramutated tumours than in other glioblastomas. Genetic analyses enabled the diagnosis of the two consanguineous childhood brain tumours as being due to a combination of PMS2 germline and POLE somatic variants, and confirmed them as bMMRD/POLE exo- disorders. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Activity inhibition and crystal polymorphism induced by active-site metal swapping.

The Arenaviridae family is one of the two RNA viral families that encode a 3'-5' exonuclease in their genome. An exonuclease domain is found in the Arenaviridae nucleoprotein and targets dsRNA specifically. This domain is directly involved in suppression of innate immunity in the host cell. Like most phosphate-processing enzymes, it requires a divalent metal ion such as Mg2+ (or Mn2+) as a cofactor to catalyse nucleotide-cleavage and nucleotide-transfer reactions. On the other hand, calcium (Ca2+) inhibits this enzymatic activity, in spite of the fact that Mg2+ and Ca2+ present comparable binding affinities and biological availabilities. Here, the molecular and structural effects of the replacement of magnesium by calcium and its inhibition mechanism for phosphodiester cleavage, an essential reaction in the viral process of innate immunity suppression, are studied. Biochemical data and high-resolution structures of the Mopeia virus exonuclease domain complexed with each ion are reported for the first time. The consequences of the ion swap for the stability of the protein, the catalytic site and the functional role of a specific metal ion in enabling the catalytic cleavage of a dsRNA substrate are outlined.

3'-Terminated Overhangs Regulate DNA Double-Strand Break Processing in Escherichia coli.

Double-strand breaks (DSBs) are lethal DNA lesions, which are repaired by homologous recombination in Escherichia coli To study DSB processing in vivo, we induced DSBs into the E. coli chromosome by γ-irradiation and measured chromosomal degradation. We show that the DNA degradation is regulated by RecA protein concentration and its rate of association with single-stranded DNA (ssDNA). RecA decreased DNA degradation in wild-type, recB, and recD strains, indicating that it is a general phenomenon in E. coli On the other hand, DNA degradation was greatly reduced and unaffected by RecA in the recB1080 mutant (which produces long overhangs) and in a strain devoid of four exonucleases that degrade a 3' tail (ssExos). 3'-5' ssExos deficiency is epistatic to RecA deficiency concerning DNA degradation, suggesting that bound RecA is shielding the 3' tail from degradation by 3'-5' ssExos. Since 3' tail preservation is common to all these situations, we infer that RecA polymerization constitutes a subset of mechanisms for preserving the integrity of 3' tails emanating from DSBs, along with 3' tail's massive length, or prevention of their degradation by inactivation of 3'-5' ssExos. Thus, we conclude that 3' overhangs are crucial in controlling the extent of DSB processing in E. coli This study suggests a regulatory mechanism for DSB processing in E. coli, wherein 3' tails impose a negative feedback loop on DSB processing reactions, specifically on helicase reloading onto dsDNA ends.

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ.

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'→5' exonuclease domain common to many replicative polymerases. In addition, Polɛ possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polɛ heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polɛ in vitro. However, similar studies of the human Polɛ heterotetramer (hPolɛ) have been limited by the difficulty of obtaining hPolɛ in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolɛ from insect host cells has allowed for isolation of greater amounts of active hPolɛ, thus enabling a more detailed kinetic comparison between hPolɛ and an active N-terminal fragment of the hPolɛ catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolɛ. We observe that the small subunits increase DNA binding by hPolɛ relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3'→5' exonuclease activity of hPolɛ is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolɛ and sway hPolɛ toward DNA synthesis rather than proofreading.