Abasic Sites Research Articles

Fluorescent detection of RNA using a base excision reporter

A fluorescent probe originally designed for the detection of abasic sites and monitoring of DNA repair has been successfully used in the detection of RNA. The strategy employs deoxyuridine in a DNA probe complementary to the target RNA; the RNA/DNA hybrid was found to be a substrate for uracil‐DNA glycosylase. After excision of uracil, rapid oxime formation between the hydroxylamine probe and the abasic site leads to a ~10‐fold increase in fluorescence in the presence of the target RNA. Additional signal can be gained by incorporating multiple deoxyuridine nucleotides through reverse transcription. Reverse transcription, base excision, and probe incorporation can be carried out concurrently in a single vessel.

From Structure Topology to Chemical Composition. XXXI. Refinement of the Crystal Structure and Chemical Formula of Selivanovaite, NaFe3+Ti4(Si2O7)2O4(H2O)4, a Murmanite-Group (Seidozerite Supergroup) TS-Block Mineral from the Lovozero Massif, Kola Peninsula, Russia

ABSTRACT Selivanovaite, ideally NaFe3+Ti4(Si2O7)2O4(H2O)4, is a murmanite-group (seidozerite supergroup) TS-block mineral from the Lovozero massif, Kola Peninsula, Russia. The crystal structure of selivanovaite was refined in space group C, a 10.524(6), b 13.815(6), c 12.213(14) Å, α 99.74(6), β 107.45(8), γ 90.15(10)°, V 1666.8(26) Å3, R1 = 21.40%. The previously given chemical analysis has been modified to better fit the crystal structure: Nb2O5 8.51, ZrO2 2.94, TiO2 31.96, SiO2 30.62, Al2O3 0.05, Fe2O3 5.08, FeO 3.23, MnO 3.36, CaO 2.14, MgO 0.75, Na2O 2.47, H2O 8.88, sum 99.99 wt.%; H2O was determined from structure-refinement results: H2O = 3.34 pfu, OH = 1.05 pfu. The empirical formula calculated on 22 O apfu is: (Na0.63Ca0.30Mn0.36)Σ1.29(Fe3+0.50Fe2+0.35)Σ0.85(Ti3.14Nb0.50Zr0.19Mg0.15Mn0.01Al0.01)Σ4.00Si3.99O22.00H7.73, Z = 4. The crystal structure of selivanovaite [basic structure type B1(MG)] is an array of TS blocks (Titanium Silicate) connected via hydrogen bonds. The TS block consists of HOH sheets (H = heteropolyhedral, O = octahedral) parallel to (001). In the O sheet, the Ti-dominant MO(1,2) sites, Na-dominant MO(3) site, and □-dominant MO(4) sites give ideally Na□Ti2pfu. In the H sheet, the Ti-dominant MH(1,2) sites, Fe3+-dominant AP(1) site, and vacant AP(2) sites give ideally Fe3+□Ti2pfu. The MH and AP(1) polyhedra and Si2O7 groups constitute the H sheet. The ideal structural formula of selivanovaite of the form AP2MH2MO4(Si2O7)2(XOM,A)4(XPM,A)4 is Fe3+□Ti2Na□Ti2(Si2O7)2O4(H2O)4. Selivanovaite is a Fe3+-bearing and OH-poor analogue of vigrishinite, ideally Zn□Ti2Na□Ti2(Si2O7)2O2O(OH)(H2O)4. Vigrishinite and selivanovaite are related by the following substitution: O(OH)–vig + H(Zn2+)vig ↔ O(O2–)sel + H(Fe3+)sel. Selivanovaite is a Fe3+-bearing and Na-poor analogue of murmanite, ideally Na2Ti2Na2Ti2(Si2O7)2O4(H2O)4. Murmanite and selivanovaite are related by the following substitution: O(Na+)mur + H(Na+2)mur ↔ O(□)sel + H(Fe3+)sel + H(□)sel. The doubling of the t1 and t2 translations of selivanovaite compared to those of murmanite is due to the ordering of Fe3+ and □ in the H sheet and Na and □ in the O sheet of selivanovaite.

Nucleophilic Thiol Proteins Bind Covalently to Abasic Sites in DNA.

In the course of studies on the enhancement of 1,2-dibromoethane-induced DNA base pair mutations by O6-alkylguanine-DNA alkyltransferase (AGT, MGMT), we discovered the facile reaction of AGT with an abasic site in DNA, leading to covalent cross-linking. The binding of AGT differs from the mechanism reported for the protein HMCES; instead it appears to involve formation of a stable thioglycoside. Facile cross-linking was also observed with the protease papain, which like AGT has a low pKa cysteine, and the tripeptide glutathione.

Breakage of cytoplasmic chromosomes by pathological DNA base excision repair.

Chromothripsis is a catastrophic mutational process that promotes tumorigenesis and causes congenital disease1-4. Chromothripsis originates from aberrations of nuclei called micronuclei or chromosome bridges5-8. These structures are associated with fragile nuclear envelopes that spontaneously rupture9,10, leading to DNA damage when chromatin is exposed to the interphase cytoplasm. Here we identify a mechanism explaining a major fraction of this DNA damage. Micronuclei accumulate large amounts of RNA-DNA hybrids, which are edited by adenine deaminases acting on RNA (ADAR enzymes) to generate deoxyinosine. Deoxyinosine is then converted into abasic sites by a DNA base excision repair (BER) glycosylase, N-methyl-purine DNA glycosylase11,12 (MPG). These abasic sites are cleaved by the BER endonuclease, apurinic/apyrimidinic endonuclease12 (APE1), creating single-stranded DNA nicks that can be converted to DNA double strand breaks by DNA replication or when closely spaced nicks occur on opposite strands13,14. This model predicts that MPG should be able to remove the deoxyinosine base from the DNA strand of RNA-DNA hybrids, which we demonstrate using purified proteins and oligonucleotide substrates. These findings identify a mechanism for fragmentation of micronuclear chromosomes, an important step in generating chromothripsis. Rather than breaking any normal chromosome, we propose that the eukaryotic cytoplasm only damages chromosomes with pre-existing defects such as the DNA base abnormality described here.

Intracellular Formation of a DNA Damage-Induced, Histone Post-Translational Modification Following Bleomycin Treatment.

Evaluating the significance of various forms of DNA damage is complicated by discoveries that some lesions inactivate repair enzymes or produce more deleterious forms of damage. Histone lysines within nucleosomes react with the commonly produced C4'-oxidized abasic site (C4-AP) to concomitantly yield an electrophilic modification (KMP) on lysine and DNA strand scission. We developed a chemoproteomic approach to identify KMP in HeLa cells. More than 60 000 KMP-modified histones are produced per cell. Using LC-MS/MS, we detected KMP at 17 of the 57 lysine residues distributed throughout the four core histone proteins. Therefore, KMP constitutes a DNA damage-induced, nonenzymatic histone post-translational modification. KMP formation suggests that downstream processes resulting from DNA damage could have ramifications on cells.

From Structure Topology to Chemical Composition. XXX. Refinement of the Crystal Structure and Chemical Formula of Shkatulkalite, Na2Nb2Na3Ti(Si2O7)2O2(FO)(H2O)4(H2O)3, a Lamprophyllite-Group (Seidozerite Supergroup) TS-Block Mineral from the Lovozero Massif, Kola Peninsula, Russia

ABSTRACT Shkatulkalite, ideally Na5TiNb2(Si2O7)2O3F(H2O)7, is a lamprophyllite-group (seidozerite supergroup) TS-block mineral from the Lovozero massif, Kola Peninsula, Russia. The crystal structure of shkatulkalite was refined as triclinic, space group P, a 5.464(1), b 7.161(1), c 15.573(1) Å, α 90.00(3), β 95.75(3), γ 90.00(3)°, V 606.3(4) Å3, R1 = 7.26%. The previously given chemical analysis has been modified to better fit the crystal structure: Nb2O5 24.15, TiO2 11.35, SiO2 27.22, BaO 1.15, SrO 2.20, MnO 1.68, CaO 0.46, K2O 0.29, Na2O 14.78, H2O 15.27, F 1.61, O = F −0.68, sum 99.48 wt.%; H2O was determined from structure-refinement results. The empirical formula was calculated on 25.27 (O + F) apfu (in accord with the crystal structure): (Na1.40Sr0.19Ba0.07K0.05)Σ1.71(Na2.86Mn0.10Ca0.07)Σ3.03(Nb1.62Ti1.27Mn0.11)Σ3Si4.03O24.52H15.11F0.75, Z = 1. The ideal structural formula is as follows: Na2Nb2Na3Ti(Si2O7)2O2(FO)(H2O)4(H2O)3. The crystal structure of shkatulkalite [basic structure type B5(LG)] is a combination of a TS (titanium-silicate) block and an I (intermediate) block. The TS block consists of HOH sheets (H-heteropolyhedral, O-octahedral). The TS block exhibits linkage and stereochemistry typical for the lamprophyllite group where Ti (+ Nb + Fe3+ + Mg) = 3 apfu. The O sheet is composed of Ti-dominant MO(1) and Na-dominant MO(2,3) octahedra. In the H sheet in shkatulkalite, Si2O7 groups link to Nb-dominant MH octahedra. The AP site occurs in the plane of the H sheet and splits into AP(1) and AP(2) sites, occupied by Na at 70% and Sr (less Ba and K) at 11%. The I block consists of H2O groups. The I block of shkatulkalite is topologically identical to those in the derivative structures of kazanskyite and nechelyustovite. The structure of the lamprophyllite-group mineral shkatulkalite has a counterpart structure in the murmanite group (Ti = 4 apfu): kolskyite, ideally Na2CaTi4(Si2O7)2O4(H2O)7 [basic structure type B7(MG)]: the two structures have TS blocks of different topology and similar I blocks, mainly occupied by H2O groups.

Study of Interaction of the PARP Family DNA-Dependent Proteins with Nucleosomes Containing DNA Intermediates of the Initial Stages of BER Process.

Reaction of (ADP-ribosyl)ation catalyzed by DNA-dependent proteins of the poly(ADP-ribose)polymerase (PARP) family, PARP1, PARP2, and PARP3, comprises the cellular response to DNA damage. These proteins are involved in the base excision repair (BER) process. Despite the extensive research, it remains unknown how PARPs are involved in the regulation of the BER process and how the roles are distributed between the DNA-dependent members of the PARP family. Here, we investigated the interaction of the PARP's family DNA-dependent proteins with nucleosome core particles containing DNA intermediates of the initial stages of BER. To do that, the nucleosomes containing damage in the vicinity of one of the DNA duplex blunt ends were reconstituted based on the Widom's Clone 603 DNA sequence. Dissociation constants of the PARP complexes with nucleosomes bearing DNA contained uracil (Native), apurine/apyrimidine site (AP site), or a single-nucleotide gap with 5'-dRp fragment (Gap) were determined. It was shown that the affinity of the proteins for the nucleosomes increased in the row: PARP3<<PARP2<PARP1; whereas the affinity of each protein for the certain damage type increased in the row: Native = AP site < Gap for PARP1 and PARP2, Gap<<<Native = AP site for PARP3. The interaction regions of each PARP protein with nucleosome were also determined by sodium borohydride cross-linking and footprinting assay. Based on the obtained and published data, the involvement pattern of the PARP1, PARP2, and PARP3 into the interaction with nucleosome particles containing DNA intermediates of the BER process was discussed.

HMCES protects immunoglobulin genes specifically from deletions during somatic hypermutation.

Somatic hypermutation (SHM) produces point mutations in immunoglobulin (Ig) genes in B cells when uracils created by the activation-induced deaminase are processed in a mutagenic manner by enzymes of the base excision repair (BER) and mismatch repair (MMR) pathways. Such uracil processing creates DNA strand breaks and is susceptible to the generation of deleterious deletions. Here, we demonstrate that the DNA repair factor HMCES strongly suppresses deletions without significantly affecting other parameters of SHM in mouse and human B cells, thereby facilitating the production of antigen-specific antibodies. The deletion-prone repair pathway suppressed by HMCES operates downstream from the uracil glycosylase UNG and is mediated by the combined action of BER factor APE2 and MMR factors MSH2, MSH6, and EXO1. HMCES's ability to shield against deletions during SHM requires its capacity to form covalent cross-links with abasic sites, in sharp contrast to its DNA end-joining role in class switch recombination but analogous to its genome-stabilizing role during DNA replication. Our findings lead to a novel model for the protection of Ig gene integrity during SHM in which abasic site cross-linking by HMCES intercedes at a critical juncture during processing of vulnerable gapped DNA intermediates by BER and MMR enzymes.

Human TDP1, APE1and TREX1 repair 3'-DNA-peptide/protein cross-links arising from abasic sites in vitro.

Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3′-phospho-α,β-unsaturated aldehyde (3′-PUA), to form unstable but long-lived Schiff base DNA–protein cross-links at 3′-DNA termini (3′-PUA–protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1’s intrinsic redox capacity, yielding a stable 3′-PUA–PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3′-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3′-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3′-PUA–protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3′-PUA–histone DPCs, but not 3′-PUA–PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3′-PUA–protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.

Label-free and highly sensitive APE1 detection based on rolling circle amplification combined with G-quadruplex

The highly sensitive detection of low-abundant apurinic/apyrimidinic endonuclease 1 (APE1) activity is of great significance for early diagnosis of disease and pathological research. Many methods for detecting APE1 based on isothermal nucleic acids amplification have been developed for improving its sensitivity. However, some of these methods have certain limitations, such as multiple reaction steps, narrow linear range, and complicated processes for fluorescent labeling. Herein, we develop a highly sensitive and label-free APE1 fluorescence detection method based on rolling circle amplification combined with G-quadruplex (RCA-G4). A hairpin probe (HP) labeled with the AP site can be recognized and cleaved by APE1, leading to the release of the primer sequence, which triggered RCA to produce long chain amplification products with a great amount of repeated sequences. The formed amplicon contains a series G-quadruplex structure, which can be combined with Thioflavin T (ThT) to produce fluorescence and achieve high sensitivity label-free detection of APE1. Benefit from the high amplification efficiency of RCA and the high fluorescence quantum yield of G-quadruplex/ThT, a detection limit as low as 1.52 × 10−6 U/mL and the linear range from 2 × 10−6 to 10 U/mL were obtained. The developed RCA-G4 method can be successfully used to detect APE1 in serum samples with a recovery from 96.3% to 105.7%. We believe that this approach is expected to play an important role in APE1-related disease research and drug development.

Biochemical characterization and mutational analysis of a mismatch glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

Mismatch glycosylase (MIG) can excise thymine and uracil from mutagenic T:G and U:G mispairs, which arise from cytosine and 5-methylcytosine deamination, respectively. Here, we present evidence that a thermostable MIG from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5 (Tb-MIG) can remove thymine and uracil from T:G and U:G mispairs at high temperature, albeit at a low efficiency for U:G mispair. The enzyme displays maximum efficiency at 70 oC - 75 °C and pH 7.0–8.0. Tb-MIG is extremely thermostable, retaining 50% activity after heating at 100 oC for 2 hrs. In addition, Tb-MIG is a bifunctional glycosylase with an AP lyase activity, then resembles the MIG from the hyperthermopilic crenarchaeon Pyrobaculum aerophilium, but contrasts with the MIG from the hyperthermopilic crenarchaeon Aeropyrum pernix. Importantly, we show that residues Y133 and D151 in Tb-MIG are essential for thymine removal, and that residues A58, N153 and R156 are involved in thymine removal. Compared with the wild-type protein, the A58D and Y133K mutants display the increased AP lyase activity, confirming the essential roles played by the correspondingly conserved Asp and Lys in endonuclease III for AP site cleavage. Overall, our work is the first biochemical characterization of a hypthermophilic euryarchaeal MIG, augmenting our understanding on archaeal MIG function.

Selective Antitumor Activity and Photocytotoxicity of Glutathione-Activated Abasic Site Trapping Agents.

Abasic (AP) sites are one of the most common DNA lesions in cells. Aldehyde-reactive alkoxyamines capture AP sites and block the activity of APE1, the enzyme responsible for initiating their repair. Blocking the APE1 repair of AP sites leads to cell death, and it is an actively investigated approach for treating cancer. However, unselective AP site capture in different cells produces side effects and limits the application of alkoxyamines in chemotherapy. Herein we take advantage of the higher glutathione (GSH) concentration in cancer cells over normal cells to develop GSH-inducible agents that selectively kill cancer cells. 2,4-Dinitrobenzenesulfonamide caged coumarin-based alkoxyamines 1 and 2 are selectively revealed by GSH to release SO2 and fluorescent coumarin-based alkoxyamines 3 and 4 that trap AP sites in cells. GSH-directed AP site trapping and SO2 release result in selective cytotoxicity (defined as IC50WI38/IC50H1299) against H1299 lung cancer cells over normal WI38 lung cells, ranging from 1.8 to 2.8 for 1 and 2. The alkylating agent methylmethanesulfonate (MMS) promotes the formation of AP sites in cells and enhances the cytotoxicity of agent 1 in a dose-dependent way. Moreover, the comet assay and γH2AX assay suggest that AP adducts form a highly toxic DNA interstrand cross-link (ICL) upon photolysis, leading to further cell death. DNA flow cytometric analysis showed that 1 promoted cell apoptosis in the early stage and induced G2/M phase cell-cycle arrest. The 2,4-dinitrobenzenesulfonamide-caged alkoxyamines exhibited selective antitumor activity and photocytotoxicity in cancer cells, illuminating their potential as GSH-directed chemotherapeutic agents.

Ultrasensitive Uracil-DNA Glycosylase Activity Assay and Its Inhibitor Screening Based on Primer Remodeling Jointly via Repair Enzyme and Polymerase.

The development of isothermal nucleic acid amplification techniques has great significance for highly sensitive biosensing in modern biology and biomedicine. A facile and robust exponential rolling circle amplification (RCA) strategy is proposed based on primer-remodeling amplification jointly via a repair enzyme and polymerase, and uracil-DNA glycosylase (UDG) is selected as a model analyte. Two kinds of complexes, complex I and complex II, are preprepared by hybridizing a circular template (CT) with a uracil-containing hairpin probe and tetrahydrofuran abasic site mimic (AP site)-embedded fluorescence-quenched probe (AFP), respectively. The target UDG specifically binds to complex I, resulting in the generation of an AP site, followed by cleavage via endonuclease IV (Endo IV) and the successive trimming of unmatched 3' terminus via phi29 DNA polymerase, thus producing a useable primer-CT complex that actuates the primary RCA. Then, numerous complex II anneal with the first-generation RCA product (RP), generating a complex II-RP assembly containing AP sites within the DNA duplex. With the aid of Endo IV and phi29, AFP, as a pre-primer in complex II, is converted into a mature primer to initiate additional rounds of RCA. So, countless AFPs are cleaved, releasing remarkably strong fluorescent signals. The biosensor is demonstrated to enable rapid and accurate detection of the UDG activity with an improved detection limit as low as 4.7 × 10-5 U·mL-1. Moreover, this biosensor is successfully applied for UDG inhibitor screening and complicated biological samples analysis. Compared to the previous exponential RCA methods, our proposed strategy offers additional advantages, including excellent stability, optional design of CT, and simplified operating steps. Therefore, this proposed strategy may create a useful and practical platform for ultrasensitive detection of low levels of analytes in clinical diagnosis and fundamental biomedicine research.

Participation of Histones in DNA Damage and Repair within Nucleosome Core Particles: Mechanism and Applications.

DNA is damaged by various endogenous and exogenous sources, leading to a diverse group of reactive intermediates that yield a complex mixture of products. The initially formed products are often metastable and can react to yield lesions that are more biologically deleterious. Mechanistic studies are frequently carried out on free DNA as the substrate. The observations do not necessarily reflect the reaction environment inside human cells where genomic DNA is condensed as chromatin in the nucleus. Chromatin is made up of monomeric structural units called nucleosomes, which are comprised of DNA wrapped around an octameric core of histone proteins (two copies each of histones H2A, H2B, H3, and H4).This account presents a summary of our work in the past decade on the mechanistic studies of DNA damage and repair in reconstituted nucleosome core particles (NCPs). A series of metastable lesions and reactive intermediates, such as abasic sites (AP), N7-methyl-2'-deoxyguanosine (MdG), and 2'-deoxyadenosin-N6-yl radical (dA•), have been independently generated in a site-specific manner in bottom-up-synthesized NCPs. Detailed mechanistic studies on these NCPs revealed that histones actively participate in DNA damage and repair processes in diverse ways. For instance, nucleophilic residues in the flexible histone N-terminal tails, such as Lys and N-terminal α-amine, react with electrophilic DNA damage and reactive intermediates. In some cases, transient intermediates are produced, leading to the promotion or suppression of damage and repair processes. In other examples, reactions with histones yield reversible or stable DNA-protein cross-links (DPCs). Histones also utilize acidic and basic residues, such as histidine and aspartic acid, to catalyze DNA strand cleavage through general acid/base catalysis. Alternatively, a Tyr in histone plays a vital role in nucleosomal DNA damage and repair via radical transfer. Finally, the reactivity discovered during the mechanistic studies has facilitated the development of new reagents and methods with applications in biotechnology.This research has enriched our knowledge of the roles of histone proteins in DNA damage and repair and their contributions to epigenetics and may have significant biological implications. The residues in histone N-terminal tails that react with DNA lesions also play pivotal roles in regulating the structure and function of chromatin, indicating that there may be cross-talk between DNA damage and repair in eukaryotic cells and epigenetic regulation. Also, in view of the biased amino acid composition of histones, these results provide hints about how the proteins have evolved to minimize their deleterious effects but maximize beneficial ones for maintaining genome integrity. Finally, previously unreported DPCs and histone post-translational modifications have been discovered through this research. The effects of these newly identified lesions on the structure and function of chromatin and their fates inside cells remain to be elucidated.

A novel ligand swing-mediated active site coordination change of human apurinic/apyrimidinic endonuclease 1: A potential cytotoxic mechanism of nickel ion in the base excision repair

Due to the main role of removing abasic sites in base-excision repair (BER), the overexpression of human apurinic/apyrimidinic endonuclease 1 (APE1) in tumor cells often induces chemotherapy resistance. APE1 contains an endogenous Mg 2+ in the active site, while the displacement by toxic Ni 2+ attenuates the effectiveness of BER. In present work, comparative molecular dynamics simulations were performed to reveal the changes in the conformation and motion-mode of APE1 44-318 after the substitution of Mg 2+ by Ni 2+ , based on the calculated force field parameters. The results show that Ni 2+ disrupts the functional opening of the loops related to DNA binding, and impairs the flexible transformation ability of metal site to cause the loss of the nucleophilic water. Our study also gives an explanation for the disorder of Mg 2+ site. This work explores the influence of Ni 2+ on the structure and function of APE1 and proposes a putative mechanism for Ni 2+ -induced BER cytotoxicity.

Base excision repair system targeting DNA adducts of trioxacarcin/LL-D49194 antibiotics for self-resistance.

Two families of DNA glycosylases (YtkR2/AlkD, AlkZ/YcaQ) have been found to remove bulky and crosslinking DNA adducts produced by bacterial natural products. Whether DNA glycosylases eliminate other types of damage formed by structurally diverse antibiotics is unknown. Here, we identify four DNA glycosylases—TxnU2, TxnU4, LldU1 and LldU5—important for biosynthesis of the aromatic polyketide antibiotics trioxacarcin A (TXNA) and LL-D49194 (LLD), and show that the enzymes provide self-resistance to the producing strains by excising the intercalated guanine adducts of TXNA and LLD. These enzymes are highly specific for TXNA/LLD-DNA lesions and have no activity toward other, less stable alkylguanines as previously described for YtkR2/AlkD and AlkZ/YcaQ. Similarly, TXNA-DNA adducts are not excised by other alkylpurine DNA glycosylases. TxnU4 and LldU1 possess unique active site motifs that provide an explanation for their tight substrate specificity. Moreover, we show that abasic (AP) sites generated from TxnU4 excision of intercalated TXNA-DNA adducts are incised by AP endonuclease less efficiently than those formed by 7mG excision. This work characterizes a distinct class of DNA glycosylase acting on intercalated DNA adducts and furthers our understanding of specific DNA repair self-resistance activities within antibiotic producers of structurally diverse, highly functionalized DNA damaging agents.

Enzymatic DNA repair cascade-driven fluorophore encoding for sensitively sensing telomerase activity in cancer cells

Telomerase is an important reverse transcriptase that is in charge of maintaining telomeres length, and the dysregulation of telomerase activity is closely associated with cancers. Accurate measurement of telomerase activity is critical to clinical diagnosis and therapeutics. Herein, by taking advantage of in vivo repair mechanisms and single-molecule detection, we demonstrate the enzymatic DNA repair cascade-driven fluorophore encoding for sensitively sensing telomerase activity in cancer cells. The presence of telomerase enables repetitive addition of (TTAGGG)n to the 3′-OH end of telomerase substrate (TS) primer to generate long telomeric products. Telomeric product then hybridizes with the apurinic (AP) probe to induce the cleavage of AP site in dsDNA by APE1, yielding a ssDNA with a biotin and an OH group at 5′ and 3′ ends and simultaneously releasing the telomeric product which in turn hybridizes with new AP probes to yield numerous ssDNAs. By adding streptavidin-coated MBs, the ssDNAs are captured and subsequently functioned as primers to initiate template-free TdT-catalyzed random incorporation of dATPs and Cy5-dATPs into 3′-OH ends, producing long poly-A chains with multiple Cy5 molecules encoding and finally forming Cy5-ssDNA-MB complexes. After magnetic separation, Cy5-ssDNA-MBs are digested by Exo I, releasing large numbers of Cy5 molecules which can be measured by single-molecule detection. This strategy exhibits excellent specificity and high sensitivity with a limit of detection (LOD) of 1 cancer cell. Moreover, it can be employed to screen inhibitors and discriminate different cancer cell lines from normal cell lines, holding enormous potential in clinical diagnosis and therapeutics.

Cleavage of Abasic Sites in DNA by an Aminoquinoxaline Compound: Augmented Cytotoxicity and DNA Damage in Combination with an Anticancer Drug Chlorambucil in Human Colorectal Carcinoma Cells.

The elevated level of endogenous oxidative DNA damage and spontaneous deamination of DNA bases in cancer cells substantially increase the abasic sites in DNA via base excision repairs (BERs). Thus, the predominant BER pathway is a favorable target for cancer therapy. Interestingly, elevated levels of glutathione (GSH) in certain cancer cells, such as colon cancer, are associated with acquired resistance to several chemotherapeutic agents, which increase the difficulty for the treatment of cancer. Here, we have reported an ideal nitro group-containing monoquinoxaline DNA intercalator (1d), which is reduced into a fluorescent quinoxaline amine (1e) in the presence of GSH; concurrently, 1e (∼100 nM concentration) selectively causes the in vitro cleavage of abasic sites in DNA. 1e also binds to the tetrahydrofuran analogue of the abasic site in the nanomolar to low micromolar range depending on the nucleotide sequence opposite to the abasic site and also induces a structural change in abasic DNA. Furthermore, the amine compound (1e) augments the response of the specific bifunctional alkylating drug chlorambucil at a much lower concentration in the human colorectal carcinoma cell (HCT-116), and their combination shows a potential strategy for targeted therapy. Alone or in combination, 1d and 1e lead to a cascade of cellular events such as induction of DNA double-stranded breaks and cell arrest at G0/G1 and G2/M phases, eventually leading to apoptotic cell death in HCT-116 cells. Hence, the outcome of this study provides a definitive approach that will help optimize the therapeutic applications for targeting the abasic site in cancer cells.

Repair-Assisted Damage Detection Reveals Biological Disparities in Prostate Cancer between African Americans and European Americans.

Simple SummaryProstate cancer is the most diagnosed cancer among men in the United States. African American men are diagnosed with and succumb to prostate cancer at higher rates than other demographic groups. Previously published works described the biological differences in prostate tumors that may contribute to poorer outcomes in African American men compared to European American men. This study was designed to explore the DNA lesion profiles found in prostate tissues. Using tissue microarrays, we found that prostate tumors from African American patients have more uracil and pyrimidine damage, elevated UNG levels, and reduced XRCC1 levels than European American tumors, which may indicate defects in the base excision repair pathway. In addition, these men had higher UMP and lower expression of folate cycle metabolites, suggesting that metabolic rewiring may also contribute to the dysregulation of base excision repair.African Americans (AA) are two times more likely to be diagnosed with and succumb to prostate cancer (PCa) compared to European Americans (EA). There is mounting evidence that biological differences in these tumors contribute to disparities in patient outcomes. Our goal was to examine the differences in DNA damage in AA and EA prostate tissues. Tissue microarrays with matched tumor-benign adjacent pairs from 77 AA and EA PCa patients were analyzed for abasic sites, oxidative lesions, crosslinks, and uracil content using the Repair Assisted Damage Detection (RADD) assay. Our analysis revealed that AA PCa, overall, have more DNA damage than EA PCa. Increased uracil and pyrimidine lesions occurred in AA tumors, while EA tumors had more oxidative lesions. AA PCa have higher levels of UMP and folate cycle metabolites than their EA counterparts. AA PCa showed higher levels of UNG, the uracil-specific glycosylase, than EA, despite uracil lesions being retained within the genome. AA patients also had lower levels of the base excision repair protein XRCC1. These results indicate dysfunction in the base excision repair pathway in AA tumors. Further, these findings reveal how metabolic rewiring in AA PCa drives biological disparities and identifies a targetable axis for cancer therapeutics.

A sensitive and selective mutation detection strategy based on non-canonical DNA structure preference of endonuclease IV

Endonuclease IV (Endo IV) is a widely used tool enzyme in biochemical research fields due to its properties in hydrolyzing the apurinic/apyrimidinic site (AP site) in the double-strand DNA (dsDNA). However, its hydrolysis activity towards the substrate with an atypical structure has not been deeply investigated yet. Here, the hydrolysis activity of Endo IV to the dsDNA substrate formed by AP-strand and partially complementary strands whose 3′-end located at different positions of the AP site was studied in detail. We found that just a single nucleotide change around the AP site could induce an apparent deceleration effect on the cleavage Endo IV in some specific structures. Benefiting from the above property, we proposed a sensitive and selective single-nucleotide variation (SNV) detection strategy without complicated probe design, auxiliary sequences or restricted experimental conditions. Furthermore, lambda exonuclease could be directly introduced to the system to recycle the target strands and enhance the detection capability. The limit of detection towards clinical-related SNV reached 0.05% mutation abundance. The detection strategy in our work expanded our horizon of properties and applications of Endo IV and inspired us to detect SNV from the perspective of dsDNA substrate structure.