Exonuclease Activity Research Articles

Measuring TREX1 and TREX2 exonuclease activities.

Three-prime Repair Exonuclease (TREX1) degrades ssDNA and dsDNA. TREX1 localizes to the perinuclear space in cells and degrades cytosolic DNA to prevent aberrant nucleic acid sensing and immune activation in humans and mice. Mutations in the TREX1 gene cause a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome, familial chilblain lupus, retinal vasculopathy with cerebral leukodystrophy, and are associated with systemic lupus erythematosus. More than 60 disease-causing TREX1 variants have been identified including dominant and recessive, missense, and frameshift mutations that map to the catalytic core region and to the C-terminal cell localization region. The TREX1-disease causing mutations affect exonuclease activity at varied levels. In this chapter, we describe methods to purify variant recombinant TREX1 enzymes and measure the exonuclease activity using ssDNA and dsDNA substrates. The relationships between TREX1 activities, types of TREX1 mutations, and TREX1-associated autoimmune diseases are considered.

Exonuclease domain mutants of yeast DIS3 display genome instability

ABSTRACTThe exosome functions to regulate the cellular transcriptome through RNA biogenesis, surveillance, and decay. Mutations in Dis3, a catalytic subunit of the RNA exosome with separable endonuclease and exonuclease activities, are linked to multiple myeloma. Here we report that a cancer-associated DIS3 allele, dis3E729K, provides evidence for DIS3 functioning in mitotic fidelity in yeast. This dis3E729K allele does not induce defects in 7S→5.8S rRNA processing, although it elicits a requirement for P-body function. While it does not significantly influence cell cycle progression alone, the allele reduces the efficiency of cell cycle arrest in strains with defects in kinetochore assembly. Finally, point mutations in the exonuclease domains of yeast Dis3 elicit genome instability phenotypes; however, these DIS3 mutations do not increase DNA damage or RNA processing defects that lead to the accumulation of polyadenylated RNA in the nucleus. These data suggest that specific DIS3 activities support mitotic fidelity in yeast.

The Nuclear RNA Exosome and Its Cofactors.

The RNA exosome is a highly conserved ribonuclease endowed with 3'-5' exonuclease and endonuclease activities. The multisubunit complex resides in both the nucleus and the cytoplasm, with varying compositions and activities between the two compartments. While the cytoplasmic exosome functions mostly in mRNA quality control pathways, the nuclear RNA exosome partakes in the 3'-end processing and complete decay of a wide variety of substrates, including virtually all types of noncoding (nc) RNAs. To handle these diverse tasks, the nuclear exosome engages with dedicated cofactors, some of which serve as activators by stimulating decay through oligoA addition and/or RNA helicase activities or, as adaptors, by recruiting RNA substrates through their RNA-binding capacities. Most nuclear exosome cofactors contain the essential RNA helicase Mtr4 (MTR4 in humans). However, apart from Mtr4, nuclear exosome cofactors have undergone significant evolutionary divergence. Here, we summarize biochemical and functional knowledge about the nuclear exosome and exemplify its cofactor variety by discussing the best understood model organisms-the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and human cells.

A Mismatch-tolerant RT-LAMP Method for Molecular Diagnosis of Highly Variable Viruses.

Loop-mediated isothermal amplification (LAMP) has been widely used in the detection of pathogens. However, there are usually numerous variants in one viral pathogen and primers employed in LAMP can hardly match all these variants. The mismatches between the primers and the viral genomes, especially those at the 3'-end of the primers, hinder LAMP reactions, leading to failure of the detection. Here, we present a mismatch-tolerant RT-LAMP protocol, which utilizes the 3'-5' exonuclease activity of the Q5 high-fidelity DNA polymerase to remove potential mismatched bases at the 3'-end of the primers during LAMP amplification. Using HIV-1 as a proof-of-principle, we showed that this protocol could represent a promising tool for accurate detection of genetically unstable viruses in laboratory, hospital and field.

Mechanistic insights into the enzymatic activity and inhibition of the replicative polymerase exonuclease domain from Mycobacterium tuberculosis

DNA replication fidelity maintains low mutation rates in bacteria. The ε-subunit of a replisome generally acts as the main proofreader during replication, using its 3′–5′ exonuclease activity to excise misincorporated bases thereby maintaining faithful replication. In Mycobacterium tuberculosis (Mtb), however, the polymerase and histidinol phosphatase (PHP) domain of the DNA polymerase DnaE1 is the primary proofreader. This domain thus maintains low mutation rates during replication and is an attractive target for drug development. Even though the structures of DnaE polymerases are available from various organisms, including Mtb, the mechanism of exonuclease activity remains elusive. In this study, we sought to unravel the mechanism and also to identify scaffolds that can specifically inhibit the exonuclease activity. To gain insight into the mode of action, we also characterized the PHP domain of the Mtb error-prone polymerase DnaE2 which shares a nearly identical active site with DnaE1-PHP. Kinetic and mutational studies allowed us to identify the critical residue involved in catalysis. Combined inhibition and computational studies also revealed a specific mode of inhibition of DnaE1-PHP by nucleoside diphosphates. Thus, this study lays the foundation for the rational design of novel inhibitors which target the Mtb replicative proofreader.

Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA.

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5′ of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3′-exonuclease activity for removing 3′-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3′-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5′ to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5′-terminus. The 3′-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3′-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

Arabidopsis Ca2+-dependent nuclease AtCaN2 plays a negative role in plant responses to salt stress

Eukaryotic nucleases are involved in processes such as DNA restriction digestion, repair, recombination, transposition, and programmed cell death (PCD). Studies on the role of nucleases have mostly focused on PCD during plant development, while the information on nucleases involved in responses to different abiotic stress conditions remains limited. Here, we identified a Ca2+-dependent nuclease, AtCaN2, in Arabidopsis thaliana and characterized its activity, expression patterns, and involvement in plant responses to salt stress. AtCaN2 showed a dual endonuclease and exonuclease activity, being able to degrade circular plasmids, RNA, single-stranded DNA, and double-stranded DNA. Expression analysis showed that AtCaN2 was strongly induced in senescent siliques and by salt stress. Overexpression of AtCaN2 decreased the plant tolerance to salt stress conditions, leading to an excessive H2O2 accumulation. However, an atcan2 mutant showed better tolerance to salt stress and a lower level of H2O2 accumulation. Moreover, the expression of several genes (AtAPX1, AtGPX8, and AtSOD1), encoding reactive oxygen species-scavenging enzymes (ascorbate peroxidase 1, glutathione peroxidase 8, and superoxide dismutase 1, respectively), was highly induced in the atcan2 mutant under salt stress conditions. In addition, salt-stress-induced cell death was increased in the AtCaN2-overexpressing transgenic plant but decreased in the atcan2 mutant. On the basis of these findings, we conclude that AtCaN2 plays a negative role in plant tolerance to salt stress. A AtCaN2 knock out could reduce ROS accumulation, decrease ROS-induced PCD, and improve overall plant tolerance.

VDJH Gene Repertoire Analysis in Multiple Myeloma (MM) Patients: Correlation with Clinical Data

IntroductionVDJH usage description in B-cell hematological malignancies has brought new insights into the clonal differentiation and clinical implications for certain pathologies, improving patients' care, although no correlation has been made in MM.We present the largest-to-date VDJH gene repertoire analysis in MM, consisting in biological and clinical data from 413 patients. This database was used to comprehensively investigate the characteristics of VDJH rearrangements: gene segment usage, somatic hypermutation (SHM), CDR3 characterization, and immunoglobulin stereotyped clusters assessment. We also investigated the potential relationship of these molecular features with the outcome.MethodsNewly diagnosed MM patients were included in the study. All were managed according to the recommendations of the GEM-PETHEMA Spanish MM group, including the inclusion of most patients in the GEM2000 and GEM2005 schemes. Monoclonal assessment was carried out with FR1 VH-JH amplification, following the BIOMED-2 methods. Moreover, 113 cases were also analyzed by NGS using the LymphoTrack IGH FR1 assay (Invivoscribe Tech, San Diego, CA).VDJH usage and mutational status were analyzed with IMGT-V-Quest. ClustalX2.0 was used to perform clustering analysis with CDR3 regions from our cohort and 1117 additional sequences obtained from IMGT/LIGM-DB corresponding to unique rearrangements from both normal and tumor human B cells. Molecular and clinical data were used to perform survival studies.ResultsThe overall VDJH detection rate was 92.5% (382/413), including 376 patients with only one detectable rearrangement, five with a biallelic rearrangement, and one with a biclonal rearrangement, also detectable by flow cytometry. Thus, 388 rearrangements where detected: 362 were productive, and 26 unproductive.Gene segments were identified in productive rearrangements. VH repertoire in MM reflected its normal counterpart, with IGHV3 being the predominant selected family, followed by IGHV4, IGHV1, IGHV2 and IGHV5. IGHD and IGHJ segment usage showed a skewed distribution, with IGHD2 and IGHD3 families accounting for 55.5% of cases, and IGHJ4 and IGHJ6 accounting for 70.8%.SHM level could be identified in 349/362 productive sequences (mean: 9.16%, SD 3.95) with statistically significant differences between certain IGHV families, namely IGHV2 was less hypermutated than IGHV1 or IGHV4. We could also collect other rearrangement features: nucleotide addition by TdT appeared in 92.5% for N1 and 88.6% for N2, with evidence of exonuclease activity in all cases. CDR3 mean length was 16±4 aminoacids (median 15, range 6-29), with preference for Gly, Ala, Asp, Tyr (~10% each one). The R/S mutation ratio was 2.39 for CDRs versus 1.74 for FWRs, showing the high influence of antigen selection over the former. No stereotyped clusters were found in our cohort.Relationship between these finding and clinical evolution was done through univariate and multivariate analyses. A shorter Progression-Free Survival (PFS) related to well-known clinical factors: presence of plasmacytomas, poor performance status or ISS stages, response to therapy, and poor-risk cytogenetics (p<0.05), as well as with gene segment selection (IGHV5, IGHD4, non IGHD2 and IGHD3), and SHM below 7%, among others (Table 1). Similar clinical data were observed for Overall Survival (OS), where the association between a short overall survival and low SHM rate (<7%) was remarkable (Figure 1).ConclusionsVDJH usage somatic hypermutation levels and CDR3 composition in multiple myeloma resembles the normal mature B cell repertoire. In contrast to Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma or Marginal Zone Lymphoma, stereotyped receptors were absent in MM, which shows no evidence of autoantigen-driven selection.Cases with SHM levels below 7% were associated with aggressive outcome that, in the same line of CLL, would reflect the expansion of more immature clones in these cases.More studies are needed to deepen in the clinical meaning of these new findings. [Display omitted] DisclosuresPuig:Celgene: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria. Ocio:Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy; Pharmamar: Consultancy; AbbVie: Consultancy; Novartis: Consultancy, Honoraria; Sanofi: Research Funding; BMS: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Mundipharma: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Array Pharmaceuticals: Research Funding. Mateos:Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Lahuerta:Amgen: Honoraria; Janssen: Honoraria; Celgene: Honoraria. San-Miguel:Celgene: Consultancy; Sanofi: Consultancy; Janssen: Consultancy; MSD: Consultancy; Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Brystol-Myers Squibb: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees. García-Sanz:Incyte: Consultancy; Amgen Inc.: Research Funding; Gilead: Research Funding; Spanish Government: Research Funding; Pharmacyclics: Research Funding; Hospira: Research Funding; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses.

T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis.

The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone for its 3′-5′ exonuclease activity or together with a 5′-3′ exonuclease for its DNA polymerase activity. Here, we present a ‘T5 exonuclease DNA assembly’ (TEDA) method that only uses a 5′-3′ exonuclease. DNA fragments with short homologous ends were treated by T5 exonuclease and then transformed into Escherichia coli to produce clone colonies. The cloning efficiency was similar to that of the commercial In-Fusion method employing a proprietary DNA polymerase, but higher than that of the Gibson method utilizing T5 exonuclease, Phusion DNA polymerase, and DNA ligase. It also assembled multiple DNA fragments and did simultaneous site-directed mutagenesis at multiple sites. The reaction mixture was simple, and each reaction used 0.04 U of T5 exonuclease that cost 0.25 US cents. The simplicity, cost effectiveness, and cloning efficiency should promote its routine use, especially for labs with a budget constraint. TEDA may trigger further development of DNA assembly methods that employ single exonucleases.

The Power of Proficiency Testing: Unraveling Single-Nucleotide Polymorphism Interference, With Potential Impact on Clinical Testing of Spinocerebellar Ataxia Type 3

The College of American Pathologists proficiency testing program has been instrumental in identifying problems in clinical testing. To describe how this program was used to identify a single-nucleotide polymorphism that affects clinical testing for spinocerebellar ataxia type 3. A proficiency testing sample with discordant results for spinocerebellar ataxia type 3 analysis was further evaluated by targeted Sanger sequencing and genotype polymerase chain reaction using multiple DNA polymerases. Of 28 laboratories responding in the spinocerebellar ataxia type 3 Proficiency Survey, 18 reported an incorrect homozygous result and 10 reported the expected heterozygous result. A heterozygous single-nucleotide polymorphism complementary to the 3' end of a published forward primer was identified in the proficiency testing sample, which may have led to allele dropout. However, this primer was used by only 3 of 18 laboratories (16%) reporting a homozygous result. A new forward primer of identical sequence, except for the 3' end being complementary to the single-nucleotide polymorphism, showed the expected heterozygous pattern. The possibility of DNA polymerase 3'-5' exonuclease activity contributing to allele dropout was investigated by testing 9 additional polymerases with and without exonuclease activity. No clear pattern emerged, but enzymes with and without 3'-5' exonuclease activity yielded both homozygous and expected heterozygous results with the published forward primer. Proactive systematic primer sequence checking is recommended because single-nucleotide polymorphism interference may result in allele dropout and impact clinical testing. Allele dropout is also influenced by other factors, including DNA polymerase exonuclease activity.

Factors Affecting the Tailing of Blunt End DNA with Fluorescent Pyrimidine dNTPs.

The transferase activity of non-proofreading DNA polymerases is a well-known phenomenon that has been utilized in cloning and sequencing applications. The non-templated addition of modified nucleotides at DNA blunt ends is a potentially useful feature of DNA polymerases that can be used for selective transformation of DNA 3' ends. In this paper, we characterized the tailing reaction at perfectly matched and mismatched duplex ends with Cy3- and Cy5-modified pyrimidine nucleotides. It was shown that the best DNA tailing substrate does not have a perfect Watson-Crick base pair at the end. Mismatched duplexes with a 3' dC were the most efficient in the Taq DNA polymerase-catalysed tailing reaction with a Cy5-modified dUTP. We further demonstrated that the arrangement of the dye residue relative to the nucleobase notably affects the outcome of the tailing reaction. A comparative study of labelled deoxycytidine and deoxyuridine nucleotides showed higher efficiency for dUTP derivatives. The non-templated addition of modified nucleotides by Taq polymerase at a duplex blunt end was generally complicated by the pyrophosphorolysis and 5' exonuclease activity of the enzyme.

Fluorometric detection of influenza viral RNA using graphene oxide

Simple and reliable detection of influenza viruses is important for timely prescription of antiviral therapy. Here, we developed a facile fluorometric system for detection of influenza subtype viral genes using graphene oxide (GO). A fluorescent DNA probe complementary to hemagglutinin gene of influenza virus is degraded by the 5′ to 3′ exonuclease activity of Taq polymerase during PCR. Upon addition of GO, the released fluorophore retains fluorescence without adsorption onto GO, whereas the intact fluorescent DNA probe is adsorbed onto GO with fluorescence quenching. Our multi well plate system can detect as low as 3.8 pg of influenza viral RNA.

The Interferon-Induced Exonuclease ISG20 Exerts Antiviral Activity through Upregulation of Type I Interferon Response Proteins.

Type I interferon (IFN)-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding the molecular basis of ISG restriction, the antiviral mechanisms of many remain unclear. The 20-kDa ISG ISG20 is a nuclear 3'-5' exonuclease with preference for single-stranded RNA (ssRNA) and has been implicated in the IFN-mediated restriction of several RNA viruses. Although the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, evidence has yet to be presented that virus inhibition in cells requires this activity. Here, we utilized a combination of an inducible, ectopic expression system and newly generated Isg20-/- mice to investigate mechanisms and consequences of ISG20-mediated restriction. Ectopically expressed ISG20 localized primarily to Cajal bodies in the nucleus and restricted replication of chikungunya and Venezuelan equine encephalitis viruses. Although restriction by ISG20 was associated with inhibition of translation of infecting genomic RNA, degradation of viral RNAs was not observed. Instead, translation inhibition of viral RNA was associated with ISG20-induced upregulation of over 100 other genes, many of which encode known antiviral effectors. ISG20 modulated the production of IFIT1, an ISG that suppresses translation of alphavirus RNAs. Consistent with this observation, the pathogenicity of IFIT1-sensitive alphaviruses was increased in Isg20-/- mice compared to that of wild-type viruses but not in cells ectopically expressing ISG20. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by regulating expression of other ISGs that inhibit translation and possibly other activities in the replication cycle.IMPORTANCE The host immune responses to infection lead to the production of type I interferon (IFN), and the upregulation of interferon-stimulated genes (ISGs) reduces virus replication and virus dissemination within a host. Ectopic expression of the interferon-induced 20-kDa exonuclease ISG20 suppressed replication of chikungunya virus and Venezuelan equine encephalitis virus, two mosquito-vectored RNA alphaviruses. Since the replication of alphavirus genomes occurs exclusively in the cytoplasm, the mechanism of nucleus-localized ISG20 inhibition of replication is unclear. In this study, we determined that ISG20 acts as a master regulator of over 100 genes, many of which are ISGs. Specifically, ISG20 upregulated IFIT1 genes and inhibited translation of the alphavirus genome. Furthermore, IFIT1-sensitive alphavirus replication was increased in Isg20-/- mice compared to the replication of wild-type viruses but not in cells ectopically expressing ISG20. We propose that ISG20 acts as an indirect regulator of RNA virus replication in the cytoplasm through the upregulation of many other ISGs.

REQUIREMENTS FOR EFFICIENT PCR CLAMPING BY LOCKED NUCLEIC ACID OLIGONUCLEOTIES FOR SIMPLE AND SENSITIVE DETECTION OF SOMATIC MUTATIONS

PCR clamping/wild-type blocking PCR with non-extendable locked nucleic acid (LNA) oligonucleotides is used for sensitive detection of somatic mutations in tumors. Various versions of the technique use different DNA polymerases and LNA oligonucleotides with and without additional phosphorothioate modifications. Here we studied requirements for successful PCR clamping with LNA oligonucleotides and Taq DNA polymerase for analysis of mutations in KRAS and BRAF genes by means of real-time PCR and Sanger sequencing. We found that addition of phosphorothioate linkages at the 5’-end of LNA oligonucleotide to protect from 5’- exonuclease activity of Taq DNA polymerase did not improve clamping. For most target sequences, efficient clamping was observed at melting temperature of LNA oligonucleotide 20‑25°C above annealing/extension temperature of the PCR with a 2-step protocol. Under such conditions, simple and sensitive detection of mutations in KRAS and BRAF genes was feasible using real-time PCR with TaqMan probes or Sanger sequencing.

HSV-1 DNA polymerase 3'-5' exonuclease-deficient mutant D368A exhibits severely reduced viral DNA synthesis and polymerase expression.

Herpesviruses, including herpes simplex virus-1, encode and express a DNA polymerase that is required for replication of their dsDNA genomes. The catalytic subunit of this enzyme contains a 3'-5' exonuclease that is involved in proofreading during replication. Although certain mutations that severely impair exonuclease activity are not lethal to the virus, it was reported that virus containing the substitution of alanine for aspartate 368 (D368A), which ablates exonuclease activity, could not be recovered, raising the possibility that this activity is essential for viral replication. To investigate this issue, we produced virus containing this mutation (D368A Pol) using a complementing cell line. D368A Pol virus was unable to form plaques on non-complementing cells. Viral DNA synthesis and polymerase activity were severely inhibited in D368A-infected cells, as was expression of the enzyme, suggesting that effects on polymerase expression rather than on exonuclease activity per se largely explain the lethal phenotype of this mutation.

Biochemical characterization of the nuclease StoNurA from the hyperthermophilic archaeon Sulfolobus tokodaii.

The DNA nuclease gene ST2109 has been cloned from the hyperthermophilic archaeon Sulfolobus tokodaii and expressed in Escherichia coli. The recombinant protein StoNurA has been purified to homogeneity by affinity chromatography and gel filtration chromatography. Biochemical analyses demonstrated that StoNurA exhibited DNA binding and 5'-3' exonuclease activities towards ssDNA and dsDNA. The temperature and pH optima of StoNurA were determined to be 65 °C and 8.0, respectively. The activity of StoNurA was found to be dependent of Mn2+, and its half-life of heat inactivation at 100 °C was 5 min. Gel filtration chromatography revealed that StoNurA could form dimers in solution. Pull-down assays also showed that StoNurA physically interacted with a DNA helicase (StoHerA). Our data suggest that NurA may play a key functional role in the processing of DNA recombinational repair.

Structure-specific endonuclease activity of SNM1A enables processing of a DNA interstrand crosslink.

DNA interstrand crosslinks (ICLs) covalently join opposing strands, blocking both replication and transcription, therefore making ICL-inducing compounds highly toxic and ideal anti-cancer agents. While incisions surrounding the ICL are required to remove damaged DNA, it is currently unclear which endonucleases are needed for this key event. SNM1A has been shown to play an important function in human ICL repair, however its suggested role has been limited to exonuclease activity and not strand incision. Here we show that SNM1A has endonuclease activity, having the ability to cleave DNA structures that arise during the initiation of ICL repair. In particular, this endonuclease activity cleaves single-stranded DNA. Given that unpaired DNA regions occur 5′ to an ICL, these findings suggest SNM1A may act as either an endonuclease and/or exonuclease during ICL repair. This finding is significant as it expands the potential role of SNM1A in ICL repair.

APURINIC/APYRIMIDINIC ENDONUCLEASE2 and ZINC FINGER DNA 3'-PHOSPHOESTERASE Play Overlapping Roles in the Maintenance of Epigenome and Genome Stability.

Base excision repair (BER) is essential for active DNA demethylation and DNA damage repair in mammals and plants. Here, we provide genetic and biochemical evidence that APURINIC/APYRIMIDINIC ENDONUCLEASE2 (APE2) plays overlapping roles with ZINC FINGER DNA 3'-PHOSPHOESTERASE (ZDP) in active DNA demethylation and DNA damage repair in Arabidopsis thaliana Simultaneous mutation of APE2 and ZDP causes DNA hypermethylation at more than 2000 loci, most of which are not hypermethylated in ape2 or zdp single mutants. The zdp and ape2 single mutants exhibit normal development, but the zdp ape2 double mutants display pleiotropic developmental defects and are supersensitive to the DNA alkylating reagent methyl methanesulfonate. The gradual accumulation of DNA lesions in the zdp ape2 seedlings is accompanied by constitutive activation of the DNA damage response and alteration of the cell cycle. Interestingly, knockout of the key DNA demethylase REPRESSOR OF SILENCING1 reduces the magnitude of DNA lesion accumulation and the DNA damage response in the zdp ape2 mutants, suggesting that a proportion of the DNA damage in the zdp ape2 mutants arises from incomplete active DNA demethylation. Lastly, we find that APE2 has 3'-phosphatase activity and strong 3'-5' exonuclease activity in vitro. Together, our results suggest that APE2 and ZDP, two BER proteins, play overlapping roles in the maintenance of epigenome and genome stability in plants.

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.

Abstract 4335: High incidence of POLE and POLD1 mutations in Chinese colorectal cancer patients identified by comprehensive genomic profiling

Abstract Introduction: The proofreading 3'-5' exonuclease activities of DNA polymerase-ϵ (POLE) and δ (POLD1) are critical for preventing mutations. POLE and POLD1 mutations can lead to high tumor mutational burdens (TMB), and predict responsiveness to immune checkpoint inhibitors (ICPI) in the absence of microsatellite instability (MSI). We therefore analyzed the genomic alterations of POLE and POLD1 in Chinese colorectal cancer (CRC) patients by comprehensive genome profiling (CGP). Method: A cohort of 123 Chinese CRC patients (pts) comprising 76 males and 47 females (median age of 60) with both FFPE tumor samples and matched peripheral blood normal control were analyzed. The majority (40%) of the patients were stage IV, 26% were stage III, 18% were stage I/II and the rest were unknown. CGP was performed with a 450-gene panel. In addition to the genomic alterations, TMB and MSI were calculated by next-generation sequencing-based algorithms. Result: CGP revealed a typical CRC mutation landscape with TP53 in 71%, APC in 70% and KRAS in 50% of the 123 Chinese pts. Fifteen percent of the pts (19/123) were MSI-H. Median TMB of all CRC pts was 11 muts/Mb, and the MSI-H subgroup had significantly higher TMB value compared to MSS subgroup (119 vs 10 muts/Mb, p-value is 1.5E-08). In total, 10% of the pts (12/123) harbored POLE and/or POLD1 known somatic mutations in the COSMIC database, including 10 POLE mutations in 8 pts and 7 POLD1 mutations in 7 pts. The 12 pts had a median age of 60 yrs (range 32-81), and 5/12 were females. Eight out of the 12 pts were associated with MSI-H. Additionally, 21 novel somatic short variants of uncertain significance (VUS) in 8 pts were detected (8/123=6.5%), and 5 pts were associated with MSI-H. Eight out of 123 Chinese CRC pts (6.5%) had POLE known mutations, which is significantly higher than 0.7% in Western population (p=9e-9). Four pts had loss-of-function (LOF) mutations. Two cases were POLE V411L associated with younger ages and the other two were truncations. All four CRC patients possess very high TMB compared to the whole cohort (305 vs 11 muts/Mb) while three of them are microsatellite stable (MSS) at stage III and IV. Conclusion: Our small cohort suggests a higher incidence of known POLE and POLD1 mutations in Chinese CRC patients and warrants further study. Our results highlight the clinical utility of comprehensive genome profiling in identifying likely responders to immune checkpoint inhibitors. Citation Format: Yinbo Chen, Sen Zhang, Wei Huang, Jun Guo, Weidong Guo, Fabo Qiu, Meihai Deng, Jingyu Cao, Samuel J. Klempner, Shun Yao, Jicheng Yao, Shou Mu, Ming Yao, Kai Wang, Weifeng Wang. High incidence of POLE and POLD1 mutations in Chinese colorectal cancer patients identified by comprehensive genomic profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4335.