Exonuclease Activity Research Articles

A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease

Human EXOG (hEXOG) is a 5′-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85 Å resolution, respectively. A Wing domain, absent in other ββα-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5′-dsDNA exonuclease activity that precisely excises a dinucleotide using an intrinsic ‘tape-measure’. The symmetrical apo hEXOG homodimer becomes asymmetrical upon binding to DNA, providing a structural basis for how substrate DNA bound to one active site allosterically regulates the activity of the other. These properties of hEXOG suggest a pathway for mitochondrial BER that provides an optimal substrate for subsequent gap-filling synthesis by DNA polymerase γ.

The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3′-5′ exonuclease that associates with a DNA replication complex

RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, i.e. in bacteria, Eukarya, and Archaea. Bacterial RecJ is a 5'-3' exonuclease and functions in DNA repair pathways by using its 5'-3' exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity but participates in the CMG complex, so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11-3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in Thermococcus kodakarensis was shown to associate with GINS and accelerate its nuclease activity and was, therefore, designated GAN (GINS-associated nuclease); however, to date, no archaeal RecJ·MCM·GINS complex has been isolated. The thermophilic archaeon Thermoplasma acidophilum has two RecJ-like proteins, designated TaRecJ1 and TaRecJ2. TaRecJ1 exhibited DNA-specific 5'-3' exonuclease activity, whereas TaRecJ2 had 3'-5' exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2·TaGINS complex stimulated activity of TaMCM (T. acidophilum MCM) helicase in vitro, and the TaRecJ2·TaMCM·TaGINS complex was also observed in vivo However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation in vitro These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya.

The kinetics of fluorescent DNA labeling using PCR with different Taq polymerases depends on the chemical structures of modified nucleotides

The kinetics of DNA labeling during PCR using six fluorescent derivatives of 2′-deoxyuridine 5′-triphosphate has been studied. These compounds differ in their chemical structure, total electric charge and the length of the linker between a dye and the C5 position of a pyrimidine base. The efficiency of the incorporation of the fluorescent derivatives into a growing DNA chain by four commercially available Taq DNA polymerases with 5′→3′ exonuclease and hot start activity has been determined using real-time PCR with a TaqMan probe and the subsequent electrophoretic analysis of the reaction products. Modified deoxyuridines with a total positive or negative charge of the chromophore were practically not incorporated by Taq polymerases during PCR. The modified deoxyuridines with a neutral charge of the chromophore were effectively incorporated into DNA. The extended length of the linker between the pyrimidine base and the chromophore led to a lower PCR inhibition and a more effective inclusion of modified nucleotides in the growing DNA chain. This fact can be explained by the reduced steric effects that were caused by the dye. As a result, the most promising combinations of fluorescently labeled nucleotide and Taq polymerase have been chosen for further use in fluorescent DNA labeling.

Structure of the family B DNA polymerase from the hyperthermophilic archaeon Pyrobaculum calidifontis.

The family B DNA polymerase from Pyrobaculum calidifontis (Pc-polymerase) consists of 783 amino acids and is magnesium-ion dependent. It has an optimal pH of 8.5, an optimal temperature of 75°C and a half-life of 4.5 h at 95°C, giving it greater thermostability than the widely used Taq DNA polymerase. The enzyme is also capable of PCR-amplifying larger DNA fragments of up to 7.5 kb in length. It was shown to have functional, error-correcting 3'-5' exonuclease activity, as do the related high-fidelity DNA polymerases from Pyrococcus furiosus, Thermococcus kodakarensis KOD1 and Thermococcus gorgonarius, which have extensive commercial applications. Pc-polymerase has a quite low sequence identity of approximately 37% to these enzymes, which, in contrast, have very high sequence identity to each other, suggesting that the P. calidifontis enzyme is distinct. Here, the structure determination of Pc-polymerase is reported, which has been refined to an R factor of 24.47% and an Rfree of 28.81% at 2.80 Å resolution. The domains of the enzyme are arranged in a circular fashion to form a disc with a narrow central channel. One face of the disc has a number of connected crevices in it, which allow the protein to bind duplex and single-stranded DNA. The central channel is thought to allow incoming nucleoside triphosphates to access the active site. The enzyme has a number of unique structural features which distinguish it from other archaeal DNA polymerases and may account for its high processivity. A model of the complex with the primer-template duplex of DNA indicates that the largest conformational change that occurs upon DNA binding is the movement of the thumb domain, which rotates by 7.6° and moves by 10.0 Å. The surface potential of the enzyme is dominated by acidic groups in the central region of the molecule, where catalytic magnesium ions bind at the polymerase and exonuclease active sites. The outer regions are richer in basic amino acids that presumably interact with the sugar-phosphate backbone of DNA. The large number of salt bridges may contribute to the high thermal stability of this enzyme.

Colorimetric and visual determination of microRNA via cycling signal amplification using T7 exonuclease

A sensitive and specific bioassay based on cyclic enzymatic amplification was developed for determination of microRNA (miRNA) by taking advantage of the exodeoxyribonuclease activity of T7 exonuclease (T7 Exo). In the presence of miRNA, DNA/RNA duplexes are formed by hybridization of miRNA and capture probes (Cp). Then, the Cp is digested to release miRNA into the next amplification cycle assisted by T7 Exo. This leads to the digestion of numerous Cp molecules. The broken Cp does no longer hybridize with hairpin probes (Hp) to unveil G-quadruplex DNAzyme (GDNAs). However, in the absence of miRNA, the Hp hybridizes with Cp to unveil GDNAs. The generated GDNAs form assemblies with hemin to form the G-quadruplex/hemin DNAzyme complex which is capable of catalyzing the oxidation of the substrate ABTS by H2O2. Upon cyclic enzymatic amplification, the output signal is reduced accordingly, this resulting in a “signal-off” signal best acquired at a wavelength of 418 nm. The lower detection limit is 0.69 pM (at an S/N ratio of 3). The assay involves efficient signal amplification, is homogeneous and isothermal, and enables visual detection. It provides a simple, rapid and sensitive platform for use in clinical diagnostics.

Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase δ mutation in human cells.

Mutations in the POLD1 and POLE genes encoding DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer (CRC) and have been found in many sporadic colorectal and endometrial tumors. Much attention has been focused on POLE exonuclease domain mutations, which occur frequently in hypermutated DNA mismatch repair (MMR)-proficient tumors and appear to be responsible for the bulk of genomic instability in these tumors. In contrast, somatic POLD1 mutations are seen less frequently and typically occur in MMR-deficient tumors. Their functional significance is often unclear. Here we demonstrate that expression of the cancer-associated POLD1-R689W allele is strongly mutagenic in human cells. The mutation rate increased synergistically when the POLD1-R689W expression was combined with a MMR defect, indicating that the mutator effect of POLD1-R689W results from a high rate of replication errors. Purified human Polδ-R689W has normal exonuclease activity, but the nucleotide selectivity of the enzyme is severely impaired, providing a mechanistic explanation for the increased mutation rate in the POLD1-R689W-expressing cells. The vast majority of mutations induced by the POLD1-R689W are GC→TA transversions and GC→AT transitions, with transversions showing a strong strand bias and a remarkable preference for polypurine/polypyrimidine sequences. The mutational specificity of the Polδ variant matches that of the hypermutated CRC cell line, HCT15, in which this variant was first identified. The results provide compelling evidence for the pathogenic role of the POLD1-R689W mutation in the development of the human tumor and emphasize the need to experimentally determine the significance of Polδ variants present in sporadic tumors.

Transcriptome-wide analysis of alternative routes for RNA substrates into the exosome complex.

The RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but it was unclear how many substrates follow each pathway in vivo. We used CRAC (UV crosslinking and analysis of cDNA) in growing cells to identify transcriptome-wide interactions of RNAs with the major nuclear exosome-cofactor Mtr4 and with individual exosome subunits (Rrp6, Csl4, Rrp41 and Rrp44) along the threaded RNA path. We compared exosome complexes lacking Rrp44 exonuclease activity, carrying a mutation in the Rrp44 S1 RNA-binding domain predicted to disfavor direct access, or with multiple mutations in Rrp41 reported to impede RNA access to the central channel in vitro. Preferential use of channel-threading was seen for mRNAs, 5S rRNA, scR1 (SRP) and aborted tRNAs transcripts. Conversely, pre-tRNAs preferentially accessed Rrp44 directly. Both routes participated in degradation and maturation of RNAPI transcripts, with hand-over during processing. Rrp41 mutations blocked substrate passage through the channel to Rrp44 only for cytoplasmic mRNAs, supporting the predicted widening of the lumen in the Rrp6-associated, nuclear complex. Many exosome substrates exhibited clear preferences for a specific path to Rrp44. Other targets showed redundancy, possibly allowing the efficient handling of highly diverse RNA-protein complexes and RNA structures. Both threading and direct access routes involve the RNA helicase Mtr4. mRNAs that are predominately nuclear or cytoplasmic exosome substrates can be distinguished in vivo.

The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C).

Chemotherapeutic nucleoside analogs, such as Ara-C, 5-Fluorouracil (5-FU) and Trifluridine (FTD), are frequently incorporated into DNA by the replicative DNA polymerases. However, it remains unclear how this incorporation kills cycling cells. There are two possibilities: Nucleoside analog triphosphates inhibit the replicative DNA polymerases, and/or nucleotide analogs mis-incorporated into genomic DNA interfere with the next round of DNA synthesis as replicative DNA polymerases recognize them as template DNA lesions, arresting synthesis. To address the first possibility, we selectively disrupted the proofreading exonuclease activity of DNA polymerase ε (Polε), the leading-strand replicative polymerase in avian DT40 and human TK6 cell lines. To address the second, we disrupted RAD18, a gene involved in translesion DNA synthesis, a mechanism that relieves stalled replication. Strikingly, POLE1exo−/− cells, but not RAD18−/− cells, were hypersensitive to Ara-C, while RAD18−/− cells were hypersensitive to FTD. gH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while gH2AX focus formation following a pulse of 5-FU and FTD was delayed to the next round of replication. Biochemical studies indicate that human proofreading-deficient Polε-exo− holoenzyme incorporates Ara-CTP, but subsequently extend from this base several times less efficiently than from intact nucleotides. Together our results suggest that Ara-C acts by blocking extension of the nascent DNA strand and is counteracted by the proofreading activity of Polε, while 5-FU and FTD are efficiently incorporated but act as replication fork blocks in the subsequent S phase, which is counteracted by translesion synthesis.

Aicardi-Goutières syndrome protein TREX1 suppresses L1 and maintains genome integrity through exonuclease-independent ORF1p depletion.

Maintaining genome integrity is important for cells and damaged DNA triggers autoimmunity. Previous studies have reported that Three-prime repair exonuclease 1(TREX1), an endogenous DNA exonuclease, prevents immune activation by depleting damaged DNA, thus preventing the development of certain autoimmune diseases. Consistently, mutations in TREX1 are linked with autoimmune diseases such as systemic lupus erythematosus, Aicardi–Goutières syndrome (AGS) and familial chilblain lupus. However, TREX1 mutants competent for DNA exonuclease activity are also linked to AGS. Here, we report a nuclease-independent involvement of TREX1 in preventing the L1 retrotransposon-induced DNA damage response. TREX1 interacted with ORF1p and altered its intracellular localization. Furthermore, TREX1 triggered ORF1p depletion and reduced the L1-mediated nicking of genomic DNA. TREX1 mutants related to AGS were deficient in inducing ORF1p depletion and could not prevent L1-mediated DNA damage. Therefore, our findings not only reveal a new mechanism for TREX1-mediated L1 suppression and uncover a new function for TREX1 in protein destabilization, but they also suggest a novel mechanism for TREX1-mediated suppression of innate immune activation through maintaining genome integrity.

Homozygosity for the WRN Helicase-Inactivating Variant, R834C, does not confer a Werner syndrome clinical phenotype

Loss-of-function mutations in the WRN helicase gene cause Werner syndrome- a progeroid syndrome with an elevated risk of cancer and other age-associated diseases. Large numbers of single nucleotide polymorphisms have been identified in WRN. We report here the organismal, cellular, and molecular phenotypes of variant rs3087425 (c. 2500C > T) that results in an arginine to cysteine substitution at residue 834 (R834C) and up to 90% reduction of WRN helicase activity. This variant is present at a high (5%) frequency in Mexico, where we identified 153 heterozygous and three homozygous individuals among 3,130 genotyped subjects. Family studies of probands identified ten additional TT homozygotes. Biochemical analysis of WRN protein purified from TT lymphoblast cell lines confirmed that the R834C substitution strongly and selectively reduces WRN helicase, but not exonuclease activity. Replication track analyses showed reduced replication fork progression in some homozygous cells following DNA replication stress. Among the thirteen TT homozygotes, we identified a previously unreported and statistically significant gender bias in favor of males (p = 0.0016), but none of the clinical findings associated with Werner syndrome. Our results indicate that WRN helicase activity alone is not rate-limiting for the development of clinical WS.

Successful Chinese German Collaborations in Historical Dresden

Successful Chinese German Collaborations in Historical Dresden

Synergistic Effects of the in cis T251I and P587L Mitochondrial DNA Polymerase γ Disease Mutations

Human mitochondrial DNA (mtDNA) polymerase γ (Pol γ) is the only polymerase known to replicate the mitochondrial genome. The Pol γ holoenzyme consists of the p140 catalytic subunit (POLG) and the p55 homodimeric accessory subunit (POLG2), which enhances binding of Pol γ to DNA and promotes processivity of the holoenzyme. Mutations within POLG impede maintenance of mtDNA and cause mitochondrial diseases. Two common POLG mutations usually found in cis in patients primarily with progressive external ophthalmoplegia generate T251I and P587L amino acid substitutions. To determine whether T251I or P587L is the primary pathogenic allele or whether both substitutions are required to cause disease, we overproduced and purified WT, T251I, P587L, and T251I + P587L double variant forms of recombinant Pol γ. Biochemical characterization of these variants revealed impaired DNA binding affinity, reduced thermostability, diminished exonuclease activity, defective catalytic activity, and compromised DNA processivity, even in the presence of the p55 accessory subunit. However, physical association with p55 was unperturbed, suggesting intersubunit affinities similar to WT. Notably, although the single mutants were similarly impaired, a dramatic synergistic effect was found for the double mutant across all parameters. In conclusion, our analyses suggest that individually both T251I and P587L substitutions functionally impair Pol γ, with greater pathogenicity predicted for the single P587L variant. Combining T251I and P587L induces extreme thermal lability and leads to synergistic nucleotide and DNA binding defects, which severely impair catalytic activity and correlate with presentation of disease in patients.

Atomistic Molecular Dynamics Simulations of Mitochondrial DNA Polymerase γ: Novel Mechanisms of Function and Pathogenesis.

DNA polymerase γ (Pol γ) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol γ replicative complex. Our simulation data show that DNA binding triggers remarkable changes in the enzyme structure, including (1) completion of the DNA-binding channel via a dynamic subdomain, which in the apo form blocks the catalytic site, (2) stabilization of the structure through the distal accessory β-subunit, and (3) formation of a putative transient replisome-binding platform in the "intrinsic processivity" subdomain of the enzyme. Our data indicate that noncatalytic mutations may disrupt replisomal interactions, thereby causing Pol γ-associated neurodegenerative disorders.

RAD51 interconnects between DNA replication, DNA repair and immunity.

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity.

A 3\u2032-5\u2032 exonuclease activity embedded in the helicase core domain of Candida albicans Pif1 helicase

3′-5′ exonucleases are frequently found to be associated to polymerases or helicases domains in the same enzyme or could function as autonomous entities. Here we uncovered that Candida albicans Pif1 (CaPif1) displays a 3′-5′ exonuclease activity besides its main helicase activity. These two latter activities appear to reside on the same polypeptide and the new exonuclease activity could be mapped to the helicase core domain. We clearly show that CaPif1 displays exclusively exonuclease activity and unambiguously establish the directionality of the exonuclease activity as the 3′-to-5′ polarity. The enzyme appears to follow the two-metal-ion driven hydrolyzing activity exhibited by most of the nucleases, as shown by its dependence of magnesium and also by the identification of aspartic residues. Interestingly, an excellent correlation could be found between the presence of the conserved residues and the exonuclease activity when testing activities on Pif1 enzymes from eight fungal organisms. In contrast to others proteins endowed with the double helicase/exonuclease functionality, CaPif1 differs in the fact that the two activities are embedded in the same helicase domain and not located on separated domains. Our findings may suggest a biochemical basis for mechanistic studies of Pif1 family helicases.

The anti/syn conformation of 8-oxo-7,8-dihydro-2′-deoxyguanosine is modulated by Bacillus subtilis PolX active site residues His255 and Asn263. Efficient processing of damaged 3′-ends

8-oxo-7,8-dihydro-2′-deoxyguanosine (8oxodG) is a major lesion resulting from oxidative stress and found in both DNA and dNTP pools. Such a lesion is usually removed from DNA by the Base Excision Repair (BER), a universally conserved DNA repair pathway. 8oxodG usually adopts the favored and promutagenic syn-conformation at the active site of DNA polymerases, allowing the base to hydrogen bonding with adenine during DNA synthesis. Here, we study the structural determinants that affect the glycosidic torsion-angle of 8oxodGTP at the catalytic active site of the family X DNA polymerase from Bacillus subtilis (PolXBs). We show that, unlike most DNA polymerases, PolXBs exhibits a similar efficiency to stabilize the anti and syn conformation of 8oxodGTP at the catalytic site. Kinetic analyses indicate that at least two conserved residues of the nucleotide binding pocket play opposite roles in the anti/syn conformation selectivity, Asn263 and His255 that favor incorporation of 8oxodGMP opposite dA and dC, respectively. In addition, the presence in PolXBs of Mn2+-dependent 3′-phosphatase and 3′-phosphodiesterase activities is also shown. Those activities rely on the catalytic center of the C-terminal Polymerase and Histidinol Phosphatase (PHP) domain of PolXBs and, together with its 3′-5′ exonuclease activity allows the enzyme to resume gap-filling after processing of damaged 3′ termini.

Single-Molecule Characterization of E. Coli Pol III Core Catalytic Activity

Antibiotic resistance is a growing health problem. Hence new antibiotics and new antibiotic targets are critically needed. Bacterial DNA polymerases are potentially attractive targets for the development of new antibiotics because, while the process of DNA replication is conserved, the structures and interactions of the proteins involved vary considerably between prokaryotes and eukaryotes. DNA polymerase III (Pol III) is the replicative DNA polymerase in E. coli and is composed of 10 subunits that tightly coordinate leading and lagging strand synthesis. The core of the polymerase (Pol III core) contains the catalytic polymerase subunit, α, the 3′ → 5′ proofreading exonuclease, e, and a subunit of unknown function, θ. Here we employ optical tweezers to characterize Pol III core activity on a single DNA molecule. We quantify the transitions between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) during Pol III core activity via constant force measurements, in which the change in extension can be used to determine the rate of polymerization or exonucleolysis at forces below the melting transition force. These experiments are performed at forces greater than the force at which dsDNA and ssDNA stretching curves cross; therefore, while polymerization is inhibited, exonucleolysis is favored by force. We show that the application of tension facilitates the inter-molecular transfer of the primer between the polymerase and exonuclease subunits of the Pol III core assembly. Here we report the dependence of these catalytic rates on template tension and find the zero-force polymerization rate of Pol III core to be 38 ± 9 nts/s. We show that the dwell times of processive events between pauses for polymerization and exonucleolysis are 0.7 ± 0.1 s and 0.3 ± 0.1 s, respectively, suggesting distinct DNA binding dynamics during the polymerase and exonuclease activities.

Real-Time Fluorescence Assays to Monitor DNA Polymerase Activities

We report fluorescence-based methods to study the polymerase and the exonuclease activities of thermostable DNA polymerases in real time. The methods are based on the use of a fluorescent reporter which become fluorescent upon binding to double-stranded DNA (method #1), the use of a quencher reporter strand displacement from a tripartite substrate (method #2) or the use of fluorescence fading of a nucleic acid stain concomitantly to DNA degradation (method #3). These methods are compatible with standard spectrofluorimeters, plate-readers or Real-Time PCR instruments. Here, we follow the efficiency of primer extension and degradation reactions by Pyrococcus abyssi thermostable family B DNA polymerases at 55°C. In addition, the size of extended products is systematically examined by gel electrophoresis followed by fluorescence visualization.These real-time methods are very sensitive, quantitative, and well suited for the screening of extension and exonuclease activity by DNA polymerases. Moreover, these assays could be used to characterize other DNA enzymes like helicases or nucleases.Based on operational properties of different DNA pols, these results could raise specific features with possible evolutionary scenario for the origin of families DNA polymerases.

Removal of ribonucleotides by p53 protein incorporated during DNA synthesis by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase frequently incorporates ribonucleotides into the proviral DNA in macrophages, but not in lymphocytes. The enzyme exerts an efficient ribonucleotide-terminated primer extension capacity. Furthermore, ribonucleotide-editing repair is attenuated in macrophages. Tumor suppressor p53 protein, displaying an intrinsic 3'→5' exonuclease activity, was found to be involved in efficient proofreading of base-base mismatches produced during DNA synthesis. As the presence of proofreading activity is cardinal for the DNA synthesis accuracy, it was of interest to assess whether p53 can serve as a trans-acting proofreader for HIV-1 reverse transcriptase during ribonucleotide incorporation. We investigated the potential involvement of cytoplasmic p53 in error correction during insertion of ribonucleotides into DNA by recombinant HIV-1 reverse transcriptase in a p53-proficient and deficient background. Primer extension reactions were carried out to elucidate the incorporation and removal of ribonucleotides. The biochemical studies suggest that p53 is involved in a ribonucleotide damage-associated repair mechanism through its capacity to remove preformed 3'-terminal ribonucleotides, to decrease ribonucleotide incorporation and to prevent the 3'-ribo-terminated primer extension during ongoing DNA synthesis by HIV-1 reverse transcriptase. A positive correlation exists between the presence of endogenous p53 and decrease in stable incorporation of ribonucleotides into DNA with p53-harboring lysates of HCT116 cells. p53, by preferential removal of purine over pyrimidine ribonucleotides, may affect the ribonucleotide mutation spectra produced by HIV-1 reverse transcriptase. The data implies that p53 can excise incorrect sugar in addition to base mispairs, thereby expanding the role of p53 in the repair of nucleic acids replication errors.

Error-Free Bypass of 7,8-dihydro-8-oxo-2'-deoxyguanosineby DNA Polymerase of Pseudomonas aeruginosa Phage PaP1.

As one of the most common forms of oxidative DNA damage, 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) generally leads to G:C to T:A mutagenesis. To study DNA replication encountering 8-oxoG by the sole DNA polymerase (Gp90) of Pseudomonas aeruginosa phage PaP1, we performed steady-state and pre-steady-state kinetic analyses of nucleotide incorporation opposite 8-oxoG by Gp90 D234A that lacks exonuclease activities on ssDNA and dsDNA substrates. Gp90 D234A could bypass 8-oxoG in an error-free manner, preferentially incorporate dCTP opposite 8-oxoG, and yield similar misincorporation frequency to unmodified G. Gp90 D234A could extend beyond C:8-oxoG or A:8-oxoG base pairs with the same efficiency. dCTP incorporation opposite G and dCTP or dATP incorporation opposite 8-oxoG showed fast burst phases. The burst of incorporation efficiency (kpol/Kd,dNTP) is decreased as dCTP:G > dCTP:8-oxoG > dATP:8-oxoG. The presence of 8-oxoG in DNA does not affect its binding to Gp90 D234A in a binary complex but it does affect it in a ternary complex with dNTP and Mg2+, and dATP misincorporation opposite 8-oxoG further weakens the binding of Gp90 D234A to DNA. This study reveals Gp90 D234A can bypass 8-oxoG in an error-free manner, providing further understanding in DNA replication encountering oxidation lesion for P.aeruginosa phage PaP1.