DNA Pol Research Articles

Novel Resistance-Associated Mutations of Thymidine Kinase and Dna Polymerase Genes of Herpes Simplex Virus Type 1 and Type 2

Studies to verify correlations between phenotypes and genotypes of herpes simplex virus (HSV) are an important tool to establish a database of resistance-associated mutations. In this study, 32 acyclovir (ACV)-resistant clinical HSV-1 and 4 ACV-resistant clinical HSV-2 isolates were examined in parallel by both phenotypic and genotypic resistance testing. Additionally, five non-viable HSV-1 strains and two non-viable HSV-2 strains with clinical resistance were included in genotypic resistance analysis. All ACV-resistant HSV isolates showed cross-resistance to brivudin and penciclovir, and were sensitive to foscarnet and cidofovir. Acyclovir resistance was assigned to frameshift and single non-synonymous mutations of the thymidine kinase (TK) gene in 32 out of 37 HSV-1 strains and in 4 out of 6 HSV-2 strains. In three HSV-1 isolates, there were resistance-associated amino acid substitutions of the DNA polymerase (pol). Six substitutions in the TK and two in the DNA pol gene could not be attributed without doubt to either ACV resistance or natural gene polymorphism. Altogether, 10 resistance-related mutations in the TK and 1 in the DNA pol gene have not been reported previously. The novel non-synonymous mutations found in this study enrich the knowledge about the genetic alterations of TK and DNA pol genes in ACV-resistant clinical HSV strains. Together with data from the literature, the findings justify the generation of a HSV database that contains resistance mutations associated with ACV resistance phenotype.

Detection of distinct human immunodeficiency virus Type 1 circulating recombinant forms in Northeast Brazil

The extraordinary genetic diversity and immune evasion of human immunodeficiency virus (HIV) pose significant challenges for vaccine development and antiretroviral therapy efficacy. The objective of this study was to characterize the molecular profile of HIV-1 epidemic in Salvador, Bahia, Brazil, determining the genetic subtypes and the presence of antiretroviral resistance mutations. HIV-1 pol DNA sequences from 57 individuals infected with HIV were obtained by PCR, followed by sequencing. The subtypes were determined by phylogenetic analyses and the intersubtype recombination was investigated by bootscanning. The pol subtypes were compared with gag and env subtypes. Antiretroviral susceptibility was evaluated through the Stanford HIV resistance Database. The subtypes frequencies were: 77.2% of subtype B, 1.8% of subtype F1, and 21.0% of BF recombinant forms. Two intergenic and three intragenic BF recombinant patterns were observed. Six (10.5%) viruses were related to CRF28/CRF29, two were related to CRF12 (3.5%), and one (1.8%) was CRF39. Fourteen (24.6%) strains carried one or more mutations associated with at least intermediate resistance: 24.6% had resistance to nucleoside reverse transcriptase inhibitors, 21.0% to non-nucleoside reverse transcriptase inhibitors, and 7% to protease inhibitors. The substitutions I54V (7.0%), M184V (14.0%), and K103N (10.5%) were the most frequent within each class of drugs. The results show a high diversity of BF genotypes and a lower prevalence of major reverse transcriptase and protease drug resistance mutations in Salvador, compared with other regions of Brazil. These findings may contribute to improve treatment strategies of patients infected with HIV-1 from this Brazilian region.

Gene Polymorphism of Thymidine Kinase and Dna Polymerase in Clinical Strains of Herpes Simplex Virus

The thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are important targets for genotypic determination of HSV resistance to antiviral drugs. The knowledge of gene polymorphism is an absolute requirement for the correct interpretation of genotypic findings. In this study, the natural polymorphism of TK and DNA pol genes was examined by DNA sequencing in 56 HSV-1 and 12 HSV-2 strains sensitive to acyclovir. In 56 HSV-1 strains, 26 different non-synonymous polymorphism-associated mutations were detected in the TK gene. To our knowledge, 8 of them have never been reported in the literature. In the TK gene of 12 HSV-2 strains, 6 polymorphism-related non-synonymous mutations were observed, whereas there was 1 novel mutation in 1 strain. The DNA pol gene of 53 HSV-1 isolates contained 47 distinct polymorphism-associated amino acid substitutions and 11 substitutions were found in the DNA pol gene of 12 HSV-2 strains. Altogether, 31 novel substitutions could be identified in the DNA pol gene of HSV-1 and 3 in HSV-2 strains. In these strains, any resistance to foscarnet was excluded. The 43 novel non-synonymous mutations enrich the knowledge about the natural genetic polymorphism of TK and DNA pol in clinical HSV strains. The findings have to be considered for genotypic analysis of HSV in case of clinical resistance.

Quantifying Ostreid Herpesvirus (OsHV-1) Genome Copies and Expression during Transmission

Understanding the pathogenic potential of a new pathogen strain or a known pathogen in a new locale is crucial for management of disease in both wild and farmed animals. The Ostreid herpesvirus-1 (OsHV-1), a known pathogen of early-life-stage Pacific oysters, Crassostrea gigas, has been associated with mortalities of juvenile oysters in many locations around the world including Tomales Bay, California. In two trials, the California OsHV-1 strain was transmitted from infected juvenile C. gigas to naïve C. gigas larvae. Survival of control larvae was high throughout both trials (97-100%) and low among those exposed to OsHV-1. No OsHV-1-exposed larvae survived to day 9 in trial 1, while trial 2 was terminated at day 7 when survival was 36.90 ± 8.66%. To assess the amount of OsHV-1 DNA present, we employed quantitative polymerase chain reaction (qPCR) assays based on the A fragment and OsHV-1 catalytic subunit of a DNA polymerase δ (DNA pol) gene. Viral genome copy numbers based on qPCR assays peaked between 3 and 5 days. To measure the presence of viable and actively transcribing virus, the DNA pol gene qPCR assay was optimized for RNA analysis after being reverse transcribed (RT-qPCR). A decline in virus gene expression was measured using RT-qPCR: relative to earlier experimental time points copy numbers were significantly lower on day 9, trial 1 (p < 0.05) and day 7, trial 2 (p < 0.05). Peaks in copies of active virus per genome occurred during two periods in trial 1 (days 1 and 5/7, p < 0.05) and one period in trial 2 (day 1, p < 0.05). Transmission electron microscopy confirmed OsHV-1 infection; herpesvirus-like nucleocapsids, capsids, and extracellular particles were visualized. We demonstrated the ability to transmit OsHV-1 from infected juvenile oysters to naïve larvae, which indicates the spread of OsHV-1 between infected hosts in the field and between commercial farms is possible. We also developed an important tool (OsHV-1-specific RT-qPCR for an active virus gene) for use in monitoring for active virus in the field and in laboratory based transmission experiments.

TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection

Both conventional and real time PCR (rt-PCR) assays based on the amplification of a 103bp fragment from the DNA polymerase (DNA pol) gene (conserved, non-structural) of Orf virus (ORFV) were developed for detection and semi-quantitation of ORFV DNA from infected cell culture and clinical samples. The latter technique was based on TaqMan chemistry. The rt-PCR assay was specific and sensitive as it could detect as low as 3.5fg or 15 copies of ORFV genomic DNA. Both intra- (0.38–1.0%) and inter-assay (0.53–2.87%) variabilities of rt-PCR were within the acceptable range meaning the high efficiency and reproducibility of the assay. The rt-PCR was applied successfully to detect ORFV DNA from suspected clinical samples. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100% and 93.5%, respectively, when compared to B2L gene based semi-nested PCR implying a wide potential of this rt-PCR for rapid field diagnosis of Orf in sheep and goats.

Exploiting the Nucleotide Substrate Specificity of Repair DNA Polymerases To Develop Novel Anticancer Agents

The genome is constantly exposed to mutations that can originate during replication or as a result of the action of both endogenous and/or exogenous damaging agents [such as reactive oxygen species (ROS), UV light, genotoxic environmental compounds, etc.]. Cells have developed a set of specialized mechanisms to counteract this mutational burden. Many cancer cells have defects in one or more DNA repair pathways, hence they rely on a narrower set of specialized DNA repair mechanisms than normal cells. Inhibiting one of these pathways in the context of an already DNA repair-deficient genetic background, will be more toxic to cancer cells than to normal cells, a concept recently exploited in cancer chemotherapy by the synthetic lethality approach. Essential to all DNA repair pathways are the DNA pols. Thus, these enzymes are being regarded as attractive targets for the development of specific inhibitors of DNA repair in cancer cells. In this review we examine the current state-of-the-art in the development of nucleotide analogs as inhibitors of repair DNA polymerases.

Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2'-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3-14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3'-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed.

Studies on Human DNA Polymerase ϵ and GINS Complex and Their Role in DNA Replication

Studies on Human DNA Polymerase ϵ and GINS Complex and Their Role in DNA Replication

Identification and characterization of acyclovir-resistant clinical HSV-1 isolates from children

Background The occurrence of herpes simplex virus (HSV) with acyclovir (ACV) resistance is a cause for concern due to the frequent use of ACV for treatment, suppressive therapy, and prophylaxis of HSV infection. Although HSV infection is prevalent among children, very little is known about the drug susceptibility of HSV circulating in this patient population. Objective To determine the status of ACV resistant HSV-1 among children. Study design A reporter cell-based HSV infection assay (mVILA) was developed to conveniently evaluate the ACV susceptibility of HSV-1 clinical strains and used to analyze 68 HSV-1 primary isolates from oral lesions in children. Results Compared with PRA, mVILA is easier to perform. Using mVILA, HSV-1 isolates C106, C153, and C174 were found completely resistant to ACV, with a greater than 100-fold increase in IC50s. Sequence analysis of thymidine kinase (TK) and DNA polymerase (DNA POL) genes identified 11 new mutations. Structural modeling of the TK and DNA POL proteins suggested structural changes that might alter their interactions with ACV and ACV triphosphate, respectively. The insertion of a single G in a seven-guanine homopolymeric repeat sequence generated a truncated TK protein in C106. Conclusion This study provides preliminary data on the ACV susceptibility status of HSV-1 in children. The prevalence rate of ACV-resistant HSV-1 in children was higher than predicted. Moreover, multiple mechanisms leading to the resistance were identified. These results suggest that new anti-herpetics with different working mechanisms should be valuable.

The evolution of HIV-1 reverse transcriptase in route to acquisition of Q151M multi-drug resistance is complex and involves mutations in multiple domains

BackgroundThe Q151M multi-drug resistance (MDR) pathway in HIV-1 reverse transcriptase (RT) confers reduced susceptibility to all nucleoside reverse transcriptase inhibitors (NRTIs) excluding tenofovir (TDF). This pathway emerges after long term failure of therapy, and is increasingly observed in the resource poor world, where antiretroviral therapy is rarely accompanied by intensive virological monitoring. In this study we examined the genotypic, phenotypic and fitness correlates associated with the development of Q151M MDR in the absence of viral load monitoring.ResultsSingle-genome sequencing (SGS) of full-length RT was carried out on sequential samples from an HIV-infected individual enrolled in ART rollout. The emergence of Q151M MDR occurred in the order A62V, V75I, and finally Q151M on the same genome at 4, 17 and 37 months after initiation of therapy, respectively. This was accompanied by a parallel cumulative acquisition of mutations at 20 other codon positions; seven of which were located in the connection subdomain. We established that fourteen of these mutations are also observed in Q151M-containing sequences submitted to the Stanford University HIV database. Phenotypic drug susceptibility testing demonstrated that the Q151M-containing RT had reduced susceptibility to all NRTIs except for TDF. RT domain-swapping of patient and wild-type RTs showed that patient-derived connection subdomains were not associated with reduced NRTI susceptibility. However, the virus expressing patient-derived Q151M RT at 37 months demonstrated ~44% replicative capacity of that at 4 months. This was further reduced to ~22% when the Q151M-containing DNA pol domain was expressed with wild-type C-terminal domain, but was then fully compensated by coexpression of the coevolved connection subdomain.ConclusionsWe demonstrate a complex interplay between drug susceptibility and replicative fitness in the acquisition Q151M MDR with serious implications for second-line regimen options. The acquisition of the Q151M pathway occurred sequentially over a long period of failing NRTI therapy, and was associated with mutations in multiple RT domains.

Resistance testing of clinical varicella-zoster virus strains

Acyclovir resistance of varicella-zoster virus (VZV) has been reported in rare cases of immunocompromised patients. In this study, the natural polymorphism of the thymidine kinase (TK) and DNA polymerase (pol) genes was examined in 51 clinical VZV isolates sensitive to acyclovir (ACV). In addition, 16 VZV strains with clinical resistance to ACV were analyzed. None of the ACV-sensitive strains of the clades 1, 3 and 5 showed gene polymorphism of the TK. By contrast, the DNA pol gene exhibited polymorphism-related substitutions as a function of the VZV clade. The novel substitutions M286I, E824Q, R984H and H1089Y were detected in strains of clades 3 and 5. In the TK gene of 7 VZV strains with clinical ACV resistance, the novel substitutions L73I, A163stop, W225R, T256M, N334stop and the deletion of nucleotides 19–223 were found to be associated most likely with resistance. In one strain showing the substitution W225R, ACV resistance could be confirmed by the viral phenotype. In the DNA pol gene, the novel amino acid substitutions T237K and A955T could be detected, but their significance remains unclear. In conclusion, the characterization of resistance using genetic analysis of the TK and DNA pol genes has to be considered the method of choice for the determination of VZV resistance to antiviral drugs. In a considerable number of patients with clinical ACV-resistant VZV infections, resistance cannot be verified by virological methods.

Disruption of mitochondrial DNA polymerase γ replication by the aldehyde adduct of DNA, M1dG

M1dG is a mutagenic adduct generated in human nuclear and mitochondrial DNA (mtDNA) by reaction of the cellular peroxidation products, malondialdehyde and base propenal, with guanine. Since M1dG is known to interfere with DNA synthesis by replicative and Y‐family DNA polymerases, we have utilized recombinant exonuclease‐deficient human DNA polymerase γ (exo− pol γ) with or without its accessory subunit p55 to investigate the effects of the adduct on mtDNA synthesis. Using primer‐templates that required exo−pol γ to extend the primer by insertion opposite the adduct position, we found that M1dG decreased exo− pol γ primer extension by 50% and prevented synthesis of a fully‐extended 35‐mer primer. A dAMP was inserted preferentially opposite M1dG, but extension of the primer past the adduct was supported only by dCMP incorporation. The insertion efficiency for exo− pol γ with either dCTP or dATP as substrates was substantially reduced by M1dG, and p55 had no effect on exo−pol γ nucleotide selection or insertion kinetics at the adduct. Since human mitochondria have no known mechanism for repair of M1dG, which is a target of nucleotide excision repair in the nucleus, our data indicate that the endogenous adduct poses a significant threat to mtDNA stability by disrupting DNA pol γ replication. (Supported by National Institutes of Health Grants GM087681 to S.D.C. and CA087819 to L.J.M, and NIH intramural funding to W.C.C.)

Local infection with oilseed rape mosaic virus promotes genetic rearrangements in systemic Arabidopsis tissue

We have previously shown that local infection of tobacco plants with tobacco mosaic virus (TMV) or oilseed rape mosaic virus (ORMV) results in a systemic increase in the homologous recombination frequency (HRF). Here, we analyzed what other changes in the genome are triggered by pathogen infection. For the analysis of HRF, mutation frequency (MF) and microsatellite instability (MI), we used three different transgenic Arabidopsis lines carrying β-glucuronidase ( GUS)-based substrates in their genome. We found that local infection of Arabidopsis with ORMV resulted in an increase of all three frequencies, albeit to differing degrees. The most prominent increase was observed in microsatellite instability. The increase in HRF was the lowest, although still statistically significant. The analysis of methylation of the 35S promoter and transgene expression showed that the greater instability of the transgene was not attributed to these changes. Strand breaks brought about a significant increase in non-treated tissues of infected plants. The expression of genes associated with various repair processes, such as KU70, RAD51, MSH2, DNA POL α and DNA POL δ, was also increased. To summarize, our data demonstrate that local ORMV infection destabilizes the genome in systemic tissues of Arabidopsis plants in various ways resulting in large rearrangements, point mutations and microsatellite instability.

Oxidative DNA Damage Bypass in Arabidopsis thaliana Requires DNA Polymerase λ and Proliferating Cell Nuclear Antigen 2

The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol λ catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol λ, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol λ, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol λ in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol λ is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes.

Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

Mutational Specificities of Brominated DNA Adducts Catalyzed by Human DNA Polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.

Escherichia coli Y family DNA polymerases

DNA damage is ubiquitous, arising from both environmental and endogenous sources. All organisms have evolved multiple pathways to respond to DNA damage and maintain genomic integrity. Escherichia coli possesses two DNA polymerases, pol IV and pol V, that are members of the Y family. These polymerases are characterized by their specialized ability to copy damaged DNA as well as their relatively low fidelity on undamaged DNA. Pol IV and pol V are regulated by the SOS response to DNA damage and by their multiple interactions with other proteins. These two Y family DNA polymerases copy DNA damaged by distinct agents. Pol IV is capable of replicating DNA containing N(2)-dG adducts, while pol V bypasses abasic sites and thymine-thymine dimers, which result from exposure to UV radiation. In addition to their roles in copying damaged DNA, the two Y family DNA polymerases in E. coli act in regulation of DNA replication and contribute to bacterial mutagenesis in response to cellular stress.

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

Genomic sequences encoding the 3' exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms.

Relaxed complex scheme suggests novel inhibitors for the lyase activity of DNA polymerase beta

DNA polymerase beta (pol β), the error-prone polymerase of base excision repair, plays a significant role in chemotherapeutic agent resistance. Its over expression reduces the efficacy of anticancer drug therapies including ionizing radiation, bleomycin, monofunctional alkylating agents and cisplatin. Small-scale studies on different types of cancer showed that pol β is mutated in approximately 30% of tumors. These mutations further lower pol β fidelity in DNA synthesis exposing the genome to serious mutations. These findings suggested pol β as a promising therapeutic target for cancer treatment. More than 60 pol β-inhibitors have been identified so far, however, most of them are either not potent or specific enough to become a drug. Here, we applied the relaxed complex scheme virtual screening (RCSVS) to allow for the full receptor flexibility in filtering the NCI diversity set, DrugBank compounds and a library of ∼9000 fragmental compounds for novel pol β inhibitors. In this procedure we screened the set of ∼12,500 compounds against an ensemble of 11 dominant-receptor structures representing the essential backbone dynamics of the 8 kDa domain of pol β. Our results predicted new compounds that can bind with higher affinity to the lyase active site compared to pamoic acid (PA), a well-known inhibitor of DNA pol β.

The Role of Replication Bypass Pathways in Dicentric Chromosome Formation in Budding Yeast

Gross chromosomal rearrangements (GCRs) are large scale changes to chromosome structure and can lead to human disease. We previously showed in Saccharomyces cerevisiae that nearby inverted repeat sequences (∼20-200 bp of homology, separated by ∼1-5 kb) frequently fuse to form unstable dicentric and acentric chromosomes. Here we analyzed inverted repeat fusion in mutants of three sets of genes. First, we show that genes in the error-free postreplication repair (PRR) pathway prevent fusion of inverted repeats, while genes in the translesion branch have no detectable role. Second, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Third, we provide evidence for a faulty template switch model by studying mutants of DNA polymerases; defects in DNA pol delta (lagging strand polymerase) and Mgs1 (a pol delta interacting protein) lead to a defect in fusion events as well as allelic recombination. Pol delta and Mgs1 may collaborate either in strand annealing and/or DNA replication involved in fusion and allelic recombination events. Fourth, by studying genes implicated in suppression of GCRs in other studies, we found that inverted repeat fusion has a profile of genetic regulation distinct from these other major forms of GCR formation.