Deoxyribose Phosphate Research Articles

Blood L-cystine levels positively related to increased risk of hypertension.

Hypertension is one component of metabolic syndrome (MetS). Here, the study evaluated hypertension-associated metabolites in relation to other MetS components. Fasting plasma samples were collected from 22 hypertensive and 63 normotensive subjects for non-targeted metabolomics. Compared with normotensive subjects, hypertensive patients were more diabetic (6.3%vs. 36.4%) and had dyslipidemia (27.0%vs. 63.6%) (both p<.05). By non-targeted metabolomics, 758 metabolites in 22 classes were identified and 56 were differentially regulated between hypertensive and normotensive groups. Amongst these 56 metabolites, receiver operating characteristic analysis showed that 14 had an area under the curve above 0.6. Multivariate-adjusted logistic regression analysis demonstrated that per one-fold increase of L-glutmatic acid, L-cystine, (9S,10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic acid, deoxyribose 5-phosphate, and falcarinolone, the odds ratios were 3.64, 4.61, 0.26, 0.26, and 0.37 for having the risk of hypertension, respectively. Of five metabolites, by Spearman's correlation analysis, only L-glutmatic acid and L-cystine levels were positively associated with systolic and diastolic blood pressure (all p<.05). Spearman's correlation analysis further revealed that L-glutmatic acid levels were positively correlated with to body mass index (BMI), fasting blood glucose, and serum triglyceride but negatively associated with HDL-c (all p<.05) whereas L-cystine levels were not related to any of these components (p ≥.13). Multivariate-adjusted linear regression analysis confirmed the positive correlation between L-cystine levels and systolic or diastolic blood pressure (β=2.66 for SBP; β=2.50 for DBP; both p<.05). In conclusion, L-cystine could be a potent metabolite for increased risk of hypertension.

Mitochondrial transcription factor A (TFAM) has 5′-deoxyribose phosphate lyase activity in vitro

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5′-deoxyribose phosphate (5′dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase β, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5′dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5′dRp residues at a rate comparable to that of DNA polymerase (pol) β, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

Postmortem Alteration of Purine Metabolism in Coronary Artery Disease.

A new approach for assisting in the diagnosis of coronary artery disease (CAD) as a cause of death is essential in cases where complete autopsy examinations are not feasible. The purine pathway has been associated with CAD patients, but the understanding of this pathway in postmortem changes needs to be explored. This study investigated the levels of blood purine metabolites in CAD after death. Heart blood samples (n = 60) were collected and divided into CAD (n = 23) and control groups (n = 37). Purine metabolites were measured via proton nuclear magnetic resonance. Guanosine triphosphate (GTP), nicotinamide adenine dinucleotide (NAD), and xanthine levels significantly decreased (p < 0.05); conversely, adenine and deoxyribose 5-phosphate levels significantly increased (p < 0.05) in the CAD group compared to the control group. Decreasing xanthine levels may serve as a marker for predicting the cause of death in CAD (AUC = 0.7). Our findings suggest that the purine pathway was interrupted by physiological processes after death, causing the metabolism of the deceased to differ from that of the living. Additionally, xanthine levels should be studied further to better understand their relationship with CAD and used as a biomarker for CAD diagnosis under decomposition and skeletonization settings.

Human 2'-Deoxynucleoside 5'-Phosphate N-Hydrolase 1: Mechanism of 2'-Deoxyuridine 5'-Monophosphate Hydrolysis.

The enzyme 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 (DNPH1) catalyzes the N-ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum kcat/KM, two groups with an average pKa of 6.4 must be deprotonated, while two groups with an average pKa of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to kcat/KM. Solvent deuterium isotope effects on kcat/KM and kcat were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.

Review on DNA Cryptography

Cryptography is the science that secures data and communication over the network by applying mathematics and logic to design strong encryption methods. In the modern era of e-business and e-commerce the protection of confidentiality, integrity and availability (CIA triad) of stored information as well as of transmitted data is very crucial. Deoxyribonucleic acid (DNA) is a genetic molecule consisting of two linked strands that wind around each other to form a double helical structure. The backbone of each strand is made of alternating deoxyribose sugar and phosphate groups. To each sugar one of four bases are attached i.e., adenine (A), cytosine (C), guanine (G) and thymine (T). DNA molecules, having the capacity to store, process and transmit information, inspires the idea of DNA cryptography. It is the rapid emerging unconventional techniques which combines the chemical characteristics of biological DNA sequences with classical cryptography to ensure non-vulnerable transmission of data. This innovative method is based on the notion of DNA computing. The methodologies of DNA cryptography are not coded mathematically; thus, it could be too secure to be cracked easily.

Human EXOG Possesses Strong AP Hydrolysis Activity: Implication on Mitochondrial DNA Base Excision Repair.

Most oxidative damage on mitochondrial DNA is corrected by the base excision repair (BER) pathway. However, the enzyme that catalyzes the rate-limiting reaction─deoxyribose phosphate (dRP) removal─in the multienzymatic reaction pathway has not been completely determined in mitochondria. Also unclear is how a logical order of enzymatic reactions is ensured. Here, we present structural and enzymatic studies showing that human mitochondrial EXOG (hEXOG) exhibits strong 5'-dRP removal ability. We show that, unlike the canonical dRP lyases that act on a single substrate, hEXOG functions on a variety of abasic sites, including 5'-dRP, its oxidized product deoxyribonolactone (dL), and the stable synthetic analogue tetrahydrofuran (THF). We determined crystal structures of hEXOG complexed with a THF-containing DNA and with a partial gapped DNA to 2.9 and 2.1 Å resolutions, respectively. The structures illustrate that hEXOG uses a controlled 5'-exonuclease activity to cleave the third phosphodiester bond away from the 5'-abasic site. This study provides a structural basis for hEXOG's broad spectrum of substrates. Further, we show that hEXOG can set the order of BER reactions by generating an ideal substrate for the subsequent reaction in BER and inhibit off-pathway reactions.

Nucleobase-modified nucleosides and nucleotides: Applications in biochemistry, synthetic biology, and drug discovery.

. DNA is often referred to as the "molecule of life" since it contains the genetic blueprint for all forms of life on this planet. The core building blocks composing DNA are deoxynucleotides. While the deoxyribose sugar and phosphate group are ubiquitous, it is the composition and spatial arrangement of the four natural nucleobases, adenine (A), cytosine (C), guanine (G), and thymine (T), that provide diversity in the coding information present in DNA. The ability of DNA to function as the genetic blueprint has historically been attributed to the formation of proper hydrogen bonding interactions made between complementary nucleobases. However, recent chemical and biochemical studies using nucleobase-modified nucleotides that contain "non-hydrogen bonding" functional groups have challenged many of the dogmatic views for the necessity of hydrogen-bonding interactions for DNA stability and function. Based on years of exciting research, this area has expanded tremendously and is thus too expansive to provide a comprehensive review on the topic. As such, this review article provides an opinion highlighting how nucleobase-modified nucleotides are being applied in diverse biomedical fields, focusing on three exciting areas of research. The first section addresses how these analogs are used as mechanistic probes for DNA polymerase activity and fidelity during replication. This section outlines the synthetic logic and medicinal chemistry approaches used to replace hydrogen-bonding functional groups to examine the contributions of shape/size, nucleobase hydrophobicity, and pi-electron interactions. The second section extends these mechanistic studies to provide insight into how nucleobase-modified nucleosides are used in synthetic biology. One example is through expansion of the genetic code in which changing the composition of DNA makes it possible to site-specifically incorporate unnatural amino acids bearing unique functional groups into enzymes and receptors. The final section describes results of pre-clinical studies using nucleobase-modified nucleosides as potential therapeutic agents against diseases such as cancer.

Regulation of futile ligation during early steps of BER in M. tuberculosis is carried out by a β-clamp-XthA-LigA tri-component complex

The Class-II AP-endonuclease (XthA) is a mycobacterial DNA base excision repair (BER) pathway enzyme that functions in the initial steps. It acts on DNA substrates that contain abasic sites to create nicks with 3′-hydroxyl (OH) and 5′-deoxyribose phosphate (5′-dRP) moieties. The NAD+-dependent DNA ligase (LigA) is the terminal player in mycobacterial BER and seals such nicks efficiently. Here, we demonstrate that the Mtbβ-clamp-MtbXthA complex that exists in the initial steps of BER engages with MtbLigA to form a novel tri-component BER complex. Size exclusion chromatography (SEC) experiments analysis show that the three proteins interact with equimolar stoichiometry. Small angle X-ray scattering (SAXS) analysis and associated studies reveal that the apo tri-component BER-complex adopts an extended conformation where MtbXthA is sandwiched between the Mtbβ-clamp and MtbLigA. The studies support that in the apo-complex MtbXthA binds subsite-I of Mtbβ-clamp through 239QLRFPKK245 motif and to MtbLigA by 104DGQPSWSGKP113 motif simultaneously. However, the complex adopts a less-extended conformation in the presence of substrate DNA, where MtbXthA interactions switch from predominantly subsite-I to subsite-II of the Mtbβ-clamp. Overall, the novel tri-component complex prevents futile ligation activity of MtbLigA on the product of MtbXthA and ensures forward progression of the pathway and productive mycobacterial BER interactions.

Molecular Basis of the Slow Growth of Mycoplasma hominis on Different Energy Sources.

Mycoplasma hominis is an opportunistic urogenital pathogen in vertebrates. It is a non-glycolytic species that produces energy via arginine degradation. Among genital mycoplasmas, M. hominis is the most commonly reported to play a role in systemic infections and can persist in the host for a long time. However, it is unclear how M. hominis proceeds under arginine limitation. The recent metabolic reconstruction of M. hominis has demonstrated its ability to catabolize deoxyribose phosphate to produce ATP. In this study, we cultivated M. hominis on two different energy sources (arginine and thymidine) and demonstrated the differences in growth rate, antibiotic sensitivity, and biofilm formation. Using label-free quantitative proteomics, we compared the proteome of M. hominis under these conditions. A total of 466 proteins were identified from M. hominis, representing approximately 85% of the predicted proteome, while the levels of 94 proteins changed significantly. As expected, we observed changes in the levels of metabolic enzymes. The energy source strongly affects the synthesis of enzymes related to RNA modifications and ribosome assembly. The translocation of lipoproteins and other membrane-associated proteins was also impaired. Our study, the first global characterization of the proteomic switching of M. hominis in arginine-deficiency media, illustrates energy source-dependent control of pathogenicity factors and can help to determine the mechanisms underlying the interaction between the growth rate and fitness of genome-reduced bacteria.

Synthetic Activity of Recombinant Whole Cell Biocatalysts Containing 2-Deoxy-D-ribose-5-phosphate Aldolase from Pectobacterium atrosepticum.

In nature 2-deoxy-D-ribose-5-phosphate aldolase (DERA) catalyses the reversible formation of 2-deoxyribose 5-phosphate from D-glyceraldehyde 3-phosphate and acetaldehyde. In addition, this enzyme can use acetaldehyde as the sole substrate, resulting in a tandem aldol reaction, yielding 2,4,6-trideoxy-D-erythro-hexapyranose, which spontaneously cyclizes. This reaction is very useful for the synthesis of the side chain of statin-type drugs used to decrease cholesterol levels in blood. One of the main challenges in the use of DERA in industrial processes, where high substrate loads are needed to achieve the desired productivity, is its inactivation by high acetaldehyde concentration. In this work, the utility of different variants of Pectobacterium atrosepticum DERA (PaDERA) as whole cell biocatalysts to synthesize 2-deoxyribose 5-phosphate and 2,4,6-trideoxy-D-erythro-hexapyranose was analysed. Under optimized conditions, E. coli BL21 (PaDERA C-His AA C49M) whole cells yields 99 % of both products. Furthermore, this enzyme is able to tolerate 500 mM acetaldehyde in a whole-cell experiment which makes it suitable for industrial applications.

Human TREX1 Repairs 3'-End DNA Lesions in Vitro.

Human three-prime repair exonuclease 1 (TREX1) is the major 3' to 5' exonuclease that functions to deplete the cytosolic DNA to prevent the autoimmune response. TREX1 is upregulated and translocates from cytoplasm to the nucleus in response to genotoxic stress, but the function of nuclear TREX1 is not well understood. Herein, we wish to report our in vitro finding that TREX1 efficiently excises 3'-phospho-α,β-unsaturated aldehyde and 3'-deoxyribose phosphate that are commonly produced as base excision repair intermediates and also from the nonenzymatic strand incision at abasic sites.

Thymidine utilisation pathway is a novel phenotypic switch of Mycoplasma hominis.

Introduction Mycoplasma hominis is a bacterium belonging to the class Mollicutes . It causes acute and chronic infections of the urogenital tract. The main features of this bacterium are an absence of cell wall and a reduced genome size (517–622 protein-encoding genes). Previously, we have isolated morphologically unknown M. hominis colonies called micro-colonies (MCs) from the serum of patients with inflammatory urogenital tract infection.HypothesisMCs are functionally different from the typical colonies (TCs) in terms of metabolism and cell division.AimTo determine the physiological differences between MCs and TCs of M. hominis and elucidate the pathways of formation and growth of MCs by a comparative proteomic analysis of these two morphological forms.MethodologyLC–MS proteomic analysis of TCs and MCs using an Ultimate 3000 RSLC nanoHPLC system connected to a QExactive Plus mass spectrometer.ResultsThe study of the proteomic profiles of M. hominis colonies allowed us to reconstruct their energy metabolism pathways. In addition to the already known pentose phosphate and arginine deamination pathways, M. hominis can utilise ribose phosphate and deoxyribose phosphate formed by nucleoside catabolism as energy sources. Comparative proteomic HPLC–MS analysis revealed that the proteomic profiles of TCs and MCs were different. We assume that MC cells preferably utilised deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Utilisation of deoxyribonucleosides is less efficient as compared with that of ribonucleosides and arginine in terms of energy production. Thymidine phosphorylase DeoA is one of the key enzymes of deoxyribonucleosides utilisation. We obtained a DeoA overexpressing mutant that exhibited a phenotype similar to that of MCs, which confirmed our hypothesis.ConclusionIn addition to the two known pathways for energy production (arginine deamination and the pentose phosphate pathway) M. hominis can use deoxyribonucleosides and ribonucleosides. MC cells demonstrate a reorganisation of energy metabolism: unlike TC cells, they preferably utilise deoxyribonucleosides, particularly thymidine, as an energy source rather than arginine or ribonucleosides. Thus MC cells enter a state of energy starvation, which helps them to survive under stress, and in particular, to be resistant to antibiotics.

K+ -Cl− -cotransport Mediates the Deoxyribose 1-phosphate-Induced Melanogenesis

The effect of deoxyribose 1-phosphate (dRP) on melanogenesis was investigated in B16 melanoma cells. dRP increased both reactive oxygen species (ROS) levels and melanin content in a dose-dependent manner in B16 cells. These effects were significantly reduced by apocynin (Apo) and diphenyleneiodonium (DPI), which are inhibitors of the NADPH oxidase (NOX). DIOA, a blocker of the K+-Cl&#8722;-cotransports (KCCs), significantly inhibited dRP-induced activation of KCCs and melanogenesis. The NOX inhibitors(Apo and DPI) also significantly blocked dRP-induced activation of KCCs. Collectively, these results suggest that dRP may activate KCCs through NOX-mediated ROS production, and in turn, induce melanogenesis in B16 cells.

Coenzyme-mediated electro-grafting for ultrasensitive electrochemical DNA biosensing

Signal amplification through the grafting of polymers has attracted increasing attention in biodetection at ultralow concentrations. Herein, an ultrasensitive peptide nucleic acid (PNA)-based electrochemical DNA biosensor is reported by using the coenzyme-mediated electro-grafting of polymers as a novel amplification method. For the capture of DNA target, PNA is used as the probe. After hybridization, reversible addition-fragmentation chain-transfer (RAFT) agents are site-specifically tethered to the deoxyribose phosphate backbone of the captured DNA targets via the carboxylate-Zr(IV)-phosphate cross-linking chemistry. Subsequently, electro-grafting of polymers through the nicotinamide adenine dinucleotide (NAD+) coenzyme-mediated electro-RAFT (NAD+-eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA) recruits numerous ferrocene redox tags for voltammetric measurement. As the electro-grafting of polymers involves simply i) the tethering of RAFT agents and ii) the NAD+-eRAFT polymerization, it is highly efficient, easy to use, and low-cost. Under optimal conditions, the voltammetric signal correlates linearly with the logarithm of DNA concentration over the range from 0.1 fM to 0.1 nM (R2 = 0.996), with a detection limit of 0.067 fM. The PNA-based electrochemical DNA biosensor can also differentiate even single base mismatch and is applicable to DNA detection in the presence of complex serum matrices, thus showing great promise in the sequence-specific detection of DNA targets at ultralow concentrations.

Urinary metabolomic analysis to identify potential markers for the diagnosis of tuberculosis and latent tuberculosis

Tuberculosis (TB) is a serious infectious disease with high infection and mortality rates. 5%–10% of the latent tuberculosis infections (LTBI) are likely to develop into active TB, and there are currently no clinical biomarkers that can distinguish between LTBI, active TB and other non-tuberculosis populations. Therefore, it is necessary to develop rapid diagnostic methods for active TB and LTBI. In this study, urinary metabolome of 30 active TB samples and the same number of LTBI and non-TB control samples were identified and analyzed by UPLC-Q Exactive MS. In total, 3744 metabolite components were obtained in ESI- mode and 4086 in ESI + mode. Orthogonal partial least square discriminant analysis (OPLS-DA) and hierarchical cluster analysis (HCA) showed that there were significant differences among LTBI, active TB and non-TB. Six differential metabolites were screened in positive and negative mode, 3-hexenoic acid, glutathione (GSH), glycochenodeoxycholate-3-sulfate, N-[4′-hydroxy-(E)-cinnamoyl]-l-aspartic acid, deoxyribose 5-phosphate and histamine. The overlapping pathways differential metabolites involved were mainly related to immune regulation and urea cycle. The results showed that the urine metabolism of TB patients was disordered and many metabolic pathways changed. Multivariate statistical analysis revealed that GSH and histamine were selected as potential molecular markers, with area under curve of receiver operating characteristic curve over 0.75. Among the multiple differential metabolites, GSH and histamine changed to varying degrees in active TB, LTBI and the non-TB control group. The levels of GSH and histamine in 48 urinary samples were measured by ELISA in validation phase, and the result in our study provided the potential for non-invasive biomarkers of TB.

5'-Deoxyribose Phosphate Lyase Activity of Apurinic/Apyrimidinic Endonuclease 1

5'-Deoxyribose Phosphate Lyase Activity of Apurinic/Apyrimidinic Endonuclease 1

The Feasibility of Studying Metabolites in PICU Multi-Organ Dysfunction Syndrome Patients over an 8-Day Course Using an Untargeted Approach.

Metabolites are generated from critical biological functions and metabolism. This pediatric study reviewed plasma metabolites in patients suffering from multi-organ dysfunction syndrome (MODS) in the pediatric intensive care unit (PICU) using an untargeted metabolomics approach. Patients meeting the criteria for MODS were screened for eligibility and consented (n = 24), and blood samples were collected at baseline, 72 h, and 8 days; control patients (n = 4) presented for routine sedation in an outpatient setting. A subset of MODS patients (n = 8) required additional support with veno-atrial extracorporeal membrane oxygenation (VA-ECMO) therapy. Metabolites from thawed blood plasma were determined from ion pairing reversed-phase liquid chromatography–mass spectrometry (LC-MS) analysis. Chromatographic peak alignment, identification, relative quantitation, and statistical and bioinformatics evaluation were performed using MAVEN and MetaboAnalyst 4.0. Metabolite analysis revealed 115 peaks per sample. From the partial least squares-discriminant analysis (PLS-DA) with variance of importance (VIP) scores above ≥2.0, 7 dynamic metabolites emerged over the three time points: tauro-chenodeoxycholic acid (TCDCA), hexose, p-hydroxybenzoate, hydroxyphenylacetic acid (HPLA), 2_3-dihydroxybenzoic acid, 2-keto-isovalerate, and deoxyribose phosphate. After Bonferroni adjustment for repeated measures, hexose and p-hydroxybenzoate were significant at one time point or more. Kendall’s tau-b test was used for internal validation of creatinine. Metabolites may be benign or significant in describing a patient’s pathophysiology and require operator interpretation.

Isomorphic building blocks for information-bearing duplexes—part 2: pyrimidine base pairs with sugar phosphate backbones

Earlier searches using DFT had identified two isomorphic H-bonded pyrimidine base pairs B1:B2 and B3:B4 as possible repeat units for macromolecular H-bonded duplexes with a capacity to store information as in DNA. This DFT study seeks to design repeat units with suitable backbones which can hold a sequence of base pairs in desired order. A deoxyribose phosphate backbone and a deoxyallopyranose phosphate backbone are proposed here. These sets of backboned nucleotide pairs are examined for their stability and their isomorphism. The results yield stable H-bonded nucleotide pairs with pairing energies of the order of those for the Watson-Crick DNA base pairs. Isomorphism is examined in the central pyrimidine base pair moiety, in the backbone torsional angles and in the overall topology. Out of three different sets of nucleotide pairs, the anionic deoxyribonucleotide pairs DA1:DA2 and DA3:DA4 emerge as showing the maximal overall isomorphism, while the pyranonucleotide pairs PyA1:PyA2 and PyA3:PyA4 are predicted as the most stable.

CUT Domains Stimulate Pol β Enzymatic Activities to Accelerate Completion of Base Excision Repair

The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase β to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol β in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5′-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.

5'-Deoxyribose Phosphate Lyase Activity of Apurinic/Apyrimidinic Endonuclease 1

One of the most common DNA lesions is the appearance of apurinic/apyrimidinic (AP-) sites. The main repair pathway for AP sites is initiated by apurinic/apyrimidinic endonuclease 1 (APE1). Upon hydrolysis of the phosphodiester bond by this enzyme, a one nucleotide gap flanked by 3'-hydroxyl and 5'-deoxyribose phosphate groups on the 5'-side of the AP site is formed. After hydrolysis of the AP site, APE1 remains associated with the product for some time. In the present work, the ability of APE1 to form a product of covalent attachment of APE1 to DNA containing a gap with a 5'-deoxyribose phosphate residue was demonstrated. In addition, it was found that while in a complex with the product of hydrolysis of the AP site, APE1 exhibits 5'-deoxyribose phosphate lyase activity, cleaving off the 5'-deoxyribose phosphate residue. The presence of lyase activity in APE1 may be important for the repair of AP sites if there is a deficiency of, or mutations in DNA polymerase β, the main enzyme that removes the 5'-deoxyribose phosphate group.