Mutant DNAs Research Articles

Temozolomide Therapy in Management of Refractory Pituitary Adenomas: A Case Series of 39 Patients

ObjectiveThe management of refractory pituitary adenomas (RPAs) presents significant challenges. This study aimed to evaluate the long-term treatment outcomes of patients with RPA managed with temozolomide (TMZ) and to identify potential biomarkers for predicting TMZ treatment response. MethodsThis retrospective case series included patients with RPA who underwent trans-sphenoidal surgery (TSS) or craniotomy at a comprehensive medical center in China between January 2014 and December 2021. Results39 patients with RPA (median age 42 years; 23 males [59%]) were treated with TMZ for a median of 9 cycles. The median follow-up was 34.4 months. Complete response (CR) was observed in 2 patients, partial response (PR) in 11 patients, stable disease (SD) in 9, progressive disease (PD) in 11, and death in 6 patients. O6-methylguanine DNA methyltransferase levels were significantly lower in patients with CR, PR, or SD compared to those with PD or mortality, with mean values of 24.2% and 58.1, respectively. MutS homologs 6 levels were significantly higher in patients with CR, PR, or SD compared to those with PD or mortality, with mean values of 64.2% and 36.9%, respectively. Patients who received concomitant TMZ and external beam radiotherapy showed a significant tumor size reduction of 178,837 mm3 (P < .001) compared to those treated with TMZ alone. ConclusionsTMZ demonstrates promising efficacy in eliciting tumor responses in patients with PRA. O6-methylguanine DNA methyltransferase and MutS homologs 6 have emerged as potential biomarkers for predicting treatment response. Furthermore, radiation with concurrent TMZ may significantly improve outcomes in patients with RPA.

The photoreactivation of 6 − 4 photoproducts in chloroplast and nuclear DNA depends on the amount of the Arabidopsis UV repair defective 3 protein

Background6 − 4 photoproducts are the second most common UV-induced DNA lesions after cyclobutane pyrimidine dimers. In plants, they are mainly repaired by photolyases in a process called photoreactivation. While pyrimidine dimers can be deleterious, leading to mutagenesis or even cell death, 6 − 4 photoproducts can activate specific signaling pathways. Therefore, their removal is particularly important, especially for plants exposed to high UV intensities due to their sessile nature. Although photoreactivation in nuclear DNA is well-known, its role in plant organelles remains unclear. In this paper we analyzed the activity and localization of GFP-tagged AtUVR3, the 6 − 4 photoproduct specific photolyase.ResultsUsing transgenic Arabidopsis with different expression levels of AtUVR3, we confirmed a positive trend between these levels and the rate of 6 − 4 photoproduct removal under blue light. Measurements of 6 − 4 photoproduct levels in chloroplast and nuclear DNA of wild type, photolyase mutants, and transgenic plants overexpressing AtUVR3 showed that the photoreactivation is the main repair pathway responsible for the removal of these lesions in both organelles. The GFP-tagged AtUVR3 was predominantly located in nuclei with a small fraction present in chloroplasts and mitochondria of transgenic Arabidopsis thaliana and Nicotiana tabacum lines. In chloroplasts, this photolyase co-localized with the nucleoid marked by plastid envelope DNA binding protein.ConclusionsPhotolyases are mainly localized in plant nuclei, with only a small fraction present in chloroplasts and mitochondria. Despite this unbalanced distribution, photoreactivation is the primary mechanism responsible for the removal of 6 − 4 photoproducts from nuclear and chloroplast DNA in adult leaves. The amount of the AtUVR3 photolyase is the limiting factor influencing the photoreactivation rate of 6 − 4 photoproducts. The efficient photoreactivation of 6 − 4 photoproducts in 35S: AtUVR3-GFP Arabidopsis and Nicotiana tabacum is a promising starting point to evaluate whether transgenic crops overproducing this photolyase are more tolerant to high UV irradiation and how they respond to other abiotic and biotic stresses under field conditions.

Hydrogen production in the Chlorella sp. DT mutants carrying heterologous electron donor ferredoxin 1 of Chlamydomonas reinhardtii

BackgroundFerredoxin 1 (Fd1) is the main electron donor to hydrogenase (HydA) for generating molecular hydrogen (H2) in green microalgae. In order to obtain an increased H2 production, the Fd1 of Chlamydomonas reinhardtii (CrFd1, encoded by crfd1) was therefore overexpressed in Chlorella sp. DT (DT) in this study. ResultsThe coding region of crfd1 was constructed into the p121-crfd1 plasmid, which was also constructed with a resistance gene to the antibiotic geneticin (G418) as a selection marker. The p121-crfd1 plasmid was transformed into DT cells by electroporation. Observation of the crfd1 gene fragment in the genomic DNA of DT-crfd1 mutants by PCR indicated that the transgene was successfully transformed. Using western blotting, the overexpressed CrFd1 protein, with a molecular weight of about 14 kDa, was found in DT-crfd1 mutants of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23. Using an in vitro assay, the H2 production of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23 mutants was about 4.4-, 5.0-, and 3.8-fold higher, respectively, than the DT wild type (DT-WT). Using an in vivo assay, the H2 production of DT-crfd1-4, DT-crfd1-22, and DT-crfd1-23 mutants was about 1.3-, 1.4-, and 1.2-fold higher, respectively, than the DT-WT. ConclusionsThe results suggested that heterologous overexpression of CrFd1 could enhance H2 production in DT-crfd1 mutants even though in vitro H2 production of DT-crfd1-22 mutant was up to 5-fold higher than the DT-WT.How to cite: Lin YJ, Chien LF. Hydrogen production in the Chlorella sp. DT mutants carrying heterologous electron donor ferredoxin 1 of Chlamydomonas reinhardtii. Electron J Biotechnol 2024;69. https://doi.org/10.1016/j.ejbt.2024.03.001.

Simultaneous Detection of Helicobacter pylori and Clarithromycin Resistance Mutations Using RAA-CRISPR/Cas13a Assay.

Infection caused by Helicobacter pylori (H. pylori) affects approximately 50% of the global population. It is a major pathogenic factor for chronic gastritis and gastric cancer. Besides, the resistance to antibiotics such as clarithromycin could reduce the eradication rate. Currently, there is an urgent need for a swift, easy to perform, and highly sensitive detection method for H. pylori and clarithromycin resistance. We used FAM/Digoxin labeled primers to amplify specific H. pylori 23S rRNA fragments by Recombinase Aided Amplification (RAA), and resistance mutations were distinguished using CRISPR/Cas13a system combined with lateral flow strip. Twenty-eight saliva samples were analyzed using qPCR, gene sequencing and this method to evaluate the detection efficiency. We developed a simultaneous detection method for H. pylori and clarithromycin resistance mutations named sensitive H. pylori easy-read dual detection (SHIELD). The results showed both A2142G and A2143G mutant DNAs causing clarithromycin resistance could be distinguished from the wild type with a concentration of 50 copies/μL, and no cross-reaction with other 5 common gastrointestinal bacteria was observed. For the detection of H. pylori in 28 saliva samples, the positive predictive value of this method was 100% (19/19) in comparison with qPCR. For detecting clarithromycin resistance, the positive predictive value of this method was 84.6% (11/13) compared with gene sequencing. SHIELD assay showed high sensitivity and specificity in detecting H. pylori and clarithromycin resistance mutations. It could be a potential measure in the rapid detection of H. pylori, large-scale screening and guiding clinical medication.

Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides.

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides. Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm3) capable of generating an electron density of greater than 1013 cm-3 was constructed to produce reactive species (N2*, N2+, O, OH, and Hα) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. This compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

Fast, sensitive, and specific multiplexed single-molecule detection of circulating tumor DNA

Circulating tumor DNA (ctDNA) analysis has emerged as a highly promising non-invasive assay for detection and monitoring of cancer. However, identification of multiple point-mutant ctDNAs, particularly at extremely low frequencies in early cancer stages, remains a significant challenge. To address this issue, we present a multiplexed ctDNA detection technique, SIMUL (single-molecule detection of multiple low-frequency mutations). SIMUL involves an unbiased preamplification of both wild-type and mutant DNAs, followed by the detection of mutant DNAs through single-molecule multicolor imaging. SIMUL enables highly sensitive and specific detection of multiple single-nucleotide mutations in a short span of time, even in the presence of 10,000-fold excess of wild-type DNA. Importantly, SIMUL can accurately measure mutant fractions due to its linear correlation between the number of single-molecule spots and the variant allele frequency. This breakthrough technique holds immense potential for clinical applications, offering significant improvements for example in early cancer detection and accurate evaluation of anticancer treatment responses.

Robust linear DNA degradation supports replication-initiation-defective mutants in Escherichia coli.

RecBCD helicase/nuclease supports replication fork progress via recombinational repair or linear DNA degradation, explaining recBC mutant synthetic lethality with replication elongation defects. Since replication initiation defects leave chromosomes without replication forks, these should be insensitive to the recBCD status. Surprisingly, we found that both Escherichia coli dnaA46(Ts) and dnaC2(Ts) initiation mutants at semi-permissive temperatures are also recBC-colethal. Interestingly, dnaA46 recBC lethality suppressors suggest underinitiation as the problem, while dnaC2 recBC suppressors signal overintiation. Using genetic and physical approaches, we studied the dnaA46 recBC synthetic lethality, for the possibility that RecBCD participates in replication initiation. Overproduced DnaA46 mutant protein interferes with growth of dnaA+ cells, while the residual viability of the dnaA46 recBC mutant depends on the auxiliary replicative helicase Rep, suggesting replication fork inhibition by the DnaA46 mutant protein. The dnaA46 mutant depends on linear DNA degradation by RecBCD, rather than on recombinational repair. At the same time, the dnaA46 defect also interacts with Holliday junction-moving defects, suggesting reversal of inhibited forks. However, in contrast to all known recBC-colethals, which fragment their chromosomes, the dnaA46 recBC mutant develops no chromosome fragmentation, indicating that its inhibited replication forks are stable. Physical measurements confirm replication inhibition in the dnaA46 mutant shifted to semi-permissive temperatures, both at the level of elongation and initiation, while RecBCD gradually restores elongation and then initiation. We propose that RecBCD-catalyzed resetting of inhibited replication forks allows replication to displace the "sticky" DnaA46(Ts) protein from the chromosomal DNA, mustering enough DnaA for new initiations.

Bacterial ribosome collision sensing by a MutS DNA repair ATPase paralogue.

Ribosome stalling during translation is detrimental to cellular fitness, but how this is sensed and elicits recycling of ribosomal subunits and quality control of associated mRNA and incomplete nascent chains is poorly understood1,2. Here we uncover Bacillus subtilis MutS2, a member of the conserved MutS family of ATPases that function in DNA mismatch repair3, as an unexpected ribosome-binding protein with an essential function in translational quality control. Cryo-electron microscopy analysis of affinity-purified native complexes shows that MutS2 functions in sensing collisions between stalled and translating ribosomes and suggests how ribosome collisions can serve as platforms to deploy downstream processes: MutS2 has an RNA endonuclease small MutS-related (SMR) domain, as well as an ATPase/clamp domain that is properly positioned to promote ribosomal subunit dissociation, which is a requirement both for ribosome recycling and for initiation of ribosome-associated protein quality control (RQC). Accordingly, MutS2 promotes nascent chain modification with alanine-tail degrons-an early step in RQC-in an ATPase domain-dependent manner. The relevance of these observations is underscored by evidence of strong co-occurrence of MutS2 and RQC genes across bacterial phyla. Overall, the findings demonstrate a deeply conserved role for ribosome collisions in mounting a complex response to the interruption of translation within open reading frames.

Increased presence of nuclear DNAJA3 and upregulation of cytosolic STAT1 and of nucleic acid sensors trigger innate immunity in the ClpP-null mouse

Mitochondrial dysfunction may activate innate immunity, e.g. upon abnormal handling of mitochondrial DNA in TFAM mutants or in altered mitophagy. Recent reports showed that also deletion of mitochondrial matrix peptidase ClpP in mice triggers transcriptional upregulation of inflammatory factors. Here, we studied ClpP-null mouse brain at two ages and mouse embryonal fibroblasts, to identify which signaling pathways are responsible, employing mass spectrometry, subcellular fractionation, immunoblots, and reverse transcriptase polymerase chain reaction. Several mitochondrial unfolded protein response factors showed accumulation and altered migration in blue-native gels, prominently the co-chaperone DNAJA3. Its mitochondrial dysregulation increased also its extra-mitochondrial abundance in the nucleus, a relevant observation given that DNAJA3 modulates innate immunity. Similar observations were made for STAT1, a putative DNAJA3 interactor. Elevated expression was observed not only for the transcription factors Stat1/2, but also for two interferon-stimulated genes (Ifi44, Gbp3). Inflammatory responses were strongest for the RLR pattern recognition receptors (Ddx58, Ifih1, Oasl2, Trim25) and several cytosolic nucleic acid sensors (Ifit1, Ifit3, Oas1b, Ifi204, Mnda). The consistent dysregulation of these factors from an early age might influence also human Perrault syndrome, where ClpP loss-of-function leads to early infertility and deafness, with subsequent widespread neurodegeneration.

Novel Escherichia coli active site dnaE alleles with altered base and sugar selectivity.

The Escherichia coli dnaE gene encodes the α‐catalytic subunit (pol IIIα) of DNA polymerase III, the cell’s main replicase. Like all high‐fidelity DNA polymerases, pol III possesses stringent base and sugar discrimination. The latter is mediated by a so‐called “steric gate” residue in the active site of the polymerase that physically clashes with the 2′‐OH of an incoming ribonucleotide. Our structural modeling data suggest that H760 is the steric gate residue in E.coli pol IIIα. To understand how H760 and the adjacent S759 residue help maintain genome stability, we generated DNA fragments in which the codons for H760 or S759 were systematically changed to the other nineteen naturally occurring amino acids and attempted to clone them into a plasmid expressing pol III core (α‐θ‐ε subunits). Of the possible 38 mutants, only nine were successfully sub‐cloned: three with substitutions at H760 and 6 with substitutions at S759. Three of the plasmid‐encoded alleles, S759C, S759N, and S759T, exhibited mild to moderate mutator activity and were moved onto the chromosome for further characterization. These studies revealed altered phenotypes regarding deoxyribonucleotide base selectivity and ribonucleotide discrimination. We believe that these are the first dnaE mutants with such phenotypes to be reported in the literature.

Coupling of the engineered DNA "mutator" to a biosensor as a new paradigm for activation of silent biosynthetic gene clusters in Streptomyces.

DNA replication fidelity in Streptomyces bacteria, prolific producers of many medically important secondary metabolites, is understudied, while in Escherichia coli it is controlled by DnaQ, the ϵ subunit of DNA polymerase III (DNA PolIII). Manipulation of dnaQ paralogues in Streptomyces lividans TK24, did not lead to increased spontaneous mutagenesis in this bacterium suggesting that S. lividans DNA PolIII uses an alternative exonuclease activity for proofreading. In Mycobacterium tuberculosis, such activity is attributed to the DnaE protein representing α subunit of DNA PolIII. Eight DnaE mutants designed based on the literature data were overexpressed in S. lividans, and recombinant strains overexpressing two of these mutants displayed markedly increased frequency of spontaneous mutagenesis (up to 1000-fold higher compared to the control). One of these ‘mutators’ was combined in S. lividans with a biosensor specific for antibiotic coelimycin, which biosynthetic gene cluster is present but not expressed in this strain. Colonies giving a positive biosensor signal appeared at a frequency of ca 10–5, and all of them were found to produce coelimycin congeners. This result confirmed that our approach can be applied for chemical- and radiation-free mutagenesis in Streptomyces leading to activation of orphan biosynthetic gene clusters and discovery of novel bioactive secondary metabolites.

A phage mechanism for selective nicking of dUMP-containing DNA

Bacteriophages (phages) have evolved efficient means to take over the machinery of the bacterial host. The molecular tools at their disposal may be applied to manipulate bacteria and to divert molecular pathways at will. Here, we describe a bacterial growth inhibitor, gene product T5.015, encoded by the T5 phage. High-throughput sequencing of genomic DNA of bacterial mutants, resistant to this inhibitor, revealed disruptive mutations in the Escherichia coli ung gene, suggesting that growth inhibition mediated by T5.015 depends on the uracil-excision activity of Ung. We validated that growth inhibition is abrogated in the absence of ung and confirmed physical binding of Ung by T5.015. In addition, biochemical assays with T5.015 and Ung indicated that T5.015 mediates endonucleolytic activity at abasic sites generated by the base-excision activity of Ung. Importantly, the growth inhibition resulting from the endonucleolytic activity is manifested by DNA replication and cell division arrest. We speculate that the phage uses this protein to selectively cause cleavage of the host DNA, which possesses more misincorporated uracils than that of the phage. This protein may also enhance phage utilization of the available resources in the infected cell, since halting replication saves nucleotides, and stopping cell division maintains both daughters of a dividing cell.

UL28 and UL33 homologs of Marek\u2019s disease virus terminase complex involved in the regulation of cleavage and packaging of viral DNA are indispensable for replication in cultured cells

Processing and packaging of herpesvirus genomic DNA is regulated by a packaging-associated terminase complex comprising of viral proteins pUL15, pUL28 and pUL33. Marek’s disease virus (MDV) homologs UL28 and UL33 showed conserved functional features with high sequence identity with the corresponding Herpes simplex virus 1 (HSV-1) homologs. As part of the investigations into the role of the UL28 and UL33 homologs of oncogenic MDV for DNA packaging and replication in cultured cells, we generated MDV mutant clones deficient in UL28 or UL33 of full-length MDV genomes. Transfection of UL28- or UL33-deleted BAC DNA into chicken embryo fibroblast (CEF) did not result either in the production of visible virus plaques, or detectable single cell infection after passaging onto fresh CEF cells. However, typical MDV plaques were detectable in CEF transfected with the DNA of revertant mutants where the deleted genes were precisely reinserted. Moreover, the replication defect of the UL28-deficient mutant was completely restored when fragment encoding the full UL28 gene was co-transfected into CEF cells. Viruses recovered from the revertant construct, as well as by the UL28 co-transfection, showed replication ability comparable with parental virus. Furthermore, the transmission electron microscopy study indicated that immature capsids were assembled without the UL28 expression, but with the loss of infectivity. Importantly, predicted three-dimensional structures of UL28 between MDV and HSV-1 suggests conserved function in virus replication. For the first time, these results revealed that both UL28 and UL33 are essential for MDV replication through regulating DNA cleavage and packaging.

PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

BackgroundThe prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs.ResultFirst, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays.ConclusionIn this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

Structural Changes in HPRT Gene of V79 Cells After Irradiation With Heavy Ions—Immediate and Delayed Effects

The radiobiological effects of accelerated ions with high charge and high energy (HZE) on mammalian cells and their propagation in time are still not sufficiently explained and attract great deal of attention. This work aims to compare the immediate and delayed effects with emphasis on the latter. As shown by our group, the dependence of mutant fraction on expression time after irradiation may have interesting, non-monotonic, character depending on LET (linear energy transfer) of the used heavy ions. We speculate that this phenomenon may occur due to the induced genomic instability. Another area of our research is the study of the DNA structural changes in these mutants induced at different expression times. Chinese hamster V79 cells were irradiated with accelerated ions 11B, 18O, 20Ne, and gamma radiation. The LET was ranging from 0.23 keV/μm of 60Co gamma rays up to 136 keV/μm of 20Ne ions. DNA of unique HPRT mutants was isolated, concentration measured, HPRT exons amplified, and analyzed at several different time points, up to about 40 days, after exposure. Over 1200 HPRT mutants were analyzed for deletions of exons and sorted into three main categories: partial deletion, PD—with deletion of one to eight exons; total deletions, TD—with all nine exons deleted; and no deletions—no change in the HPRT structure observed. In general, the number of samples with partial deletion was increasing with LET of the used radiation, suggesting that higher energy deposition to the cell nucleus is more likely to cause larger structural changes. In the case of total deletions, increase in their number with LET was observed up to LET ∼115 keV/μm followed by a sharp decrease. The samples were also analyzed for the distribution of deletions, in particular exons at various expression times, the so-called mutational patterns. Hypothesis of the mechanisms behind observed phenomena is given, and possible implications for further research are discussed.

Genome copy number regulates inclusion expansion, septation, and infectious developmental form conversion in Chlamydia trachomatis.

DNA replication is essential for the growth and development of Chlamydia trachomatis, however it is unclear how this process contributes to and is controlled by the pathogen's biphasic lifecycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature sensitive dnaE mutant (dnaEts ) that exhibits a ∼100-fold reduction in genome copy number at the non-permissive temperature (40°C), but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in dnaEts at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. dnaEts formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernable peptidoglycan and polymerized MreB, the major cell division organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion reversed when dnaEts was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE also restored dnaEts genome replication and inclusion expansion at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe's replicative and infectious forms.SIGNIFICANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and non-infectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen's infectious cycle has not been characterized. We show that genome replication is dispensable for EB to RB conversion, but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe's biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.

Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis.

Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics-even below this breakpoint-is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA, which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M. tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M. tuberculosis survival during isoniazid treatment via reduced expression of katG, the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M. tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic.

Liquid biopsy using extracellular vesicle-derived DNA in lung adenocarcinoma.

Blood liquid biopsy has emerged as a way of overcoming the clinical limitations of repeat biopsy by testing for the presence of acquired resistance mutations to therapeutic agents. Despite its merits of repeatability and non-invasiveness, this method is currently only used as a supplemental test due to a relatively low sensitivity rate of 50%–60%, and cannot replace tissue biopsy. The circulating tumor DNAs used in blood liquid biopsies are passive products of fragmented DNA with a short half-life released following tumor cell death; the low sensitivity seen with liquid blood biopsy results from this instability, which makes increasing the sensitivity of this test fundamentally difficult. Extracellular vesicles (EVs) are ideal carriers of cancer biomarkers, as cancer cells secret an abundance of EVs, and the contents of tumor cell-originated EVs reflect the molecular and genetic composition of parental cells. In addition, EV-derived DNAs (EV DNAs) consist of large-sized genomic DNAs and tumor-specific oncogenic mutant DNAs. For these reasons, liquid biopsy using EV DNA has the potential to overcome issues arising from tissue shortages associated with small biopsies, which are often seen in lung cancer patients, and the biopsy product can be used in other diagnostic methods, such as epidermal growth factor receptor (EGFR) mutation testing and next-generation sequencing (NGS). A higher sensitivity can be achieved when EV DNAs obtained from bronchoalveolar lavage fluid (BALF) are used rather than those from blood. BALF, when obtained close to the tumor site, is a promising liquid biopsy tool, as it enables the gathering of both cellular and non-cellular fractions of the tumor microenvironment, and provides increased diagnostic sensitivity when compared to blood.

Large-scale contractions of Friedreich’s ataxia GAA repeats in yeast occur during DNA replication due to their triplex-forming ability

Friedreich's ataxia (FRDA) is a human hereditary disease caused by the presence of expanded (GAA)n repeats in the first intron of the FXN gene [V. Campuzano et al., Science 271, 1423-1427 (1996)]. In somatic tissues of FRDA patients, (GAA)n repeat tracts are highly unstable, with contractions more common than expansions [R. Sharma et al., Hum. Mol. Genet. 11, 2175-2187 (2002)]. Here we describe an experimental system to characterize GAA repeat contractions in yeast and to conduct a genetic analysis of this process. We found that large-scale contraction is a one-step process, resulting in a median loss of ∼60 triplet repeats. Our genetic analysis revealed that contractions occur during DNA replication, rather than by various DNA repair pathways. Repeats contract in the course of lagging-strand synthesis: The processivity subunit of DNA polymerase δ, Pol32, and the catalytic domain of Rev1, a translesion polymerase, act together in the same pathway to counteract contractions. Accumulation of single-stranded DNA (ssDNA) in the lagging-strand template greatly increases the probability that (GAA)n repeats contract, which in turn promotes repeat instability in rfa1, rad27, and dna2 mutants. Finally, by comparing contraction rates for homopurine-homopyrimidine repeats differing in their mirror symmetry, we found that contractions depend on a repeat's triplex-forming ability. We propose that accumulation of ssDNA in the lagging-strand template fosters the formation of a triplex between the nascent and fold-back template strands of the repeat. Occasional jumps of DNA polymerase through this triplex hurdle, result in repeat contractions in the nascent lagging strand.

Inactivation of Genes Encoding MutL and MutS Proteins Influences Adhesion and Biofilm Formation by Neisseria gonorrhoeae.

Neisseria gonorrhoeae is an etiological agent of gonorrhea, which remains a global health problem. This bacterium possesses MutL and MutS DNA repair proteins encoded by mutL and mutS genes, whose inactivation causes a mutator phenotype. We have demonstrated the differential gene expression in N. gonorrhoeae mutL and mutS mutants using DNA microarrays. A subset of differentially expressed genes encodes proteins that can influence adhesion and biofilm formation. Compared to the wild-type strain, N. gonorrhoeae mutL and mutS mutants formed denser biofilms with increased biofilm-associated biomass on the abiotic surface. The N. gonorrhoeae mutS::km, but not the mutL mutant, was also more adherent and invasive to human epithelial cells. Further, during infection of epithelial cells with N. gonorrhoeae mutS::km, the expression of some bacterial genes encoding proteins that can influence gonococcal adhesion was changed compared with their expression in cells infected with the wild-type gonococcus, as well as of human genes’ encoding receptors utilized by N. gonorrhoeae (CD46, CEACAM 1, HSPG 2). Thus, deficiency in the mutS gene resulting in increased mutation frequency in singular organisms can be beneficial in populations because these mutants can be a source of features linked to microbial fitness.