Bursicon regulates wing expansion via PKA in the oriental fruit fly, Bactrocera dorsalis.

BACKGROUND: Bursicon is a heterodimeric neuropeptide that is involved in many physiological activities such as cuticle tanning, wing expansion, reproduction and immunity in insects. In this study, the role of bursicon in the wing expansion was investigated in Bactrocera dorsalis, an important invasive insect pest in agriculture. RESULTS: The cDNA sequences and deduced amino acids of bursicon genes (named BdBurs-α and BdBurs-ß) were determined, and two proteins typically contained 11 cysteine residues in conserved positions that were highly conserved in other insect species. The spatiotemporal expressions of bursicon genes showed that higher expression occurred at the pupal, early adult stage and ovaries, and lower expression at the late larval stage and in wing tissue (8-day-old pupae). Dysfunction of bursicon genes by dsRNA microinjection into 5-day-old pupae reduced PKA (a downstream component of the bursicon pathway) activity and resulted in malformed adult wings. PKA inhibitor injection into 5-day-old pupae also resulted in similar phenotypes. Hematoxylin & eosin staining of the adult wing showed that RNAi and PKA inhibitor treatment reduced the thickness of the wing cuticle, which wing cuticle thickness were ≈50% thinner than in the control. Furthermore, the expression of hedgehog (Bdhh) (one of 10 tested genes related to wing development) was significantly upregulated after RNAi and PKA inhibitor application. CONCLUSION: The results indicate that bursicon plays a crucial role in the wing expansion of B. dorsalis, suggesting bursicon genes have potential to be the targets for B. dorsalis control. © 2023 Society of Chemical Industry.

Pif1 family helicases promote mutation avoidance during DNA replication.

Pif1 family 5' → 3' DNA helicases are important for replication fork progression and genome stability. The budding yeast Saccharomyces cerevisiae encodes two Pif1 family helicases, Rrm3 and Pif1, both of which are multi-functional. Here we describe novel functions for Rrm3 in promoting mutation avoidance during DNA replication. We show that loss of RRM3 results in elevated spontaneous mutations made by DNA polymerases Pols ϵ and δ, which are subject to DNA mismatch repair. The absence of RRM3 also causes higher mutagenesis by the fourth B-family DNA polymerase Pol ζ. By genome-wide analysis, we show that the mutational consequences due to loss of RRM3 vary depending on the genomic locus. Rrm3 promotes the accuracy of DNA replication by Pols ϵ and δ across the genome, and it is particularly important for preventing Pol ζ-dependent mutagenesis at tRNA genes. In addition, mutation avoidance by Rrm3 depends on its helicase activity, and Pif1 serves as a backup for Rrm3 in suppressing mutagenesis. We present evidence that the sole human Pif1 family helicase in human cells likely also promotes replication fidelity, suggesting that a role for Pif1 family helicases in mutation avoidance may be evolutionarily conserved, a possible underlying mechanism for its potential tumor-suppressor function.

Extrinsic proofreading.

The high fidelity of replication of the nuclear DNA genome in eukaryotes involves three processes. Correct rather than incorrect dNTPs are almost always incorporated by the three major replicases, DNA polymerases α, δ and ε. When an incorrect base is occasionally inserted, the latter Pols δ and ε also have a 3 ´ to 5 ´ exonuclease activity that can remove the mismatch to allow correct DNA synthesis to proceed. Lastly, rare mismatches that escape proofreading activity and are present in newly replicated DNA can be removed by DNA mismatch repair. In this review, we consider evidence supporting the hypothesis that the second mechanism, proofreading, can operate in two different ways. Primer terminal mismatches made by either Pol δ or Pol ε can be 'intrinsically' proofread. This mechanism occurs by direct transfer of a misinserted base made at the polymerase active site to the exonuclease active site that is located a short distance away. Intrinsic proofreading allows mismatch excision without intervening enzyme dissociation. Alternatively, considerable evidence suggests that mismatches made by any of the three replicases can also be proofread by 'extrinsic' proofreading by Pol δ. Extrinsic proofreading occurs when a mismatch made by any of the three replicases is initially abandoned, thereby allowing the exonuclease active site of Pol δ to bind directly to and remove the mismatch before replication continues. Here we review the evidence that extrinsic proofreading significantly enhances the fidelity of nuclear DNA replication, and we then briefly consider the implications of this process for evolution and disease.

Transcriptome Analysis Reveals Molecular Underpinnings of Common Carp (Cyprinus carpio) Under Hypoxia Stress.

As an essential environmental factor that affects the economic benefits of aquaculture, hypoxia is one of the urgent problems to be solved in the aquaculture fish breeding industry. Common carp (Cyprinus carpio) is a critical economic fish in China, and at present, there are many breeding strains of common carp with different character advantages in China, including Hebao red carp (C. carpio var wuyuanesis) and Songpu mirror carp (C. carpio var specularis). Even if the environmental adaptation of common carp is generally strong, the genetic background of hypoxia tolerance in different strains of common carp is unclear yet. This study tested the hypoxia tolerance of Songpu minor carp, Hebao red carp, and their hybrid F1 population by an acute hypoxia treatment. Muscle and liver tissues were used for transcriptome sequencing analysis to identify the key factors for hypoxia tolerance and explore the potential genetic mechanism for breeding high hypoxia tolerance in common carp. The comparative transcriptomic analysis revealed abundant hypoxia response-related genes and their differential regulation mechanism in these two tissues of different common carp strains under acute hypoxia, including immune response, cellular stress response, HIFs (hypoxia-inducible factors), MAP kinase, iron ion binding, and heme binding. Our findings will facilitate future investigation on the hypoxia response mechanism and provide a solid theoretical basis for breeding projects in common carp.

CYP314A1-dependent 20-hydroxyecdysone biosynthesis is involved in regulating the development of pupal diapause and energy metabolism in the Chinese citrus fruit fly, Bactrocera minax.

BACKGROUND: Diapause is an environmentally preprogrammed period of arrested development, and characterized by metabolic depression that can occur during any development stage of insect. The insect steroid hormone 20-hydroxyecdysone (20E), is converted from ecdysone by the cytochrome P450 enzyme shade (CYP314A1), and it exerts a potent effect on the induction and maintenance of diapause in obligatory diapause insects. However, the regulatory mechanism of 20E in obligatory diapause development remains unclear. In this study, the function of 20E in the pupal diapause of Bactrocera minax was investigated. RESULTS: We determined the expression pattern of Halloween P450 genes from larval to adult B. minax, and found differential expression of CYP314A1 from other P450 genes, with a high level in larvae and a low level in pupae. Dysfunction of CYP314A1 by dsCYP314A1 microinjection in third-instar larvae caused significant larval mortality or abnormal pupae. Compared with dsGFP and DEPC-water, dsCYP314A1-injected larvae had significantly reduced 20E titer and altered energy metabolism, and many individuals failed to pupate. Exogenous 20E microinjected into late third-instar larvae or 20E fed to early third-instar larvae both caused similar energy metabolism changes. The 20E-treated larvae of B. minax had reduced total lipids and increased amounts of trehalose and glycogen. Furthermore, 20E-treated diapause individuals showed rapid pupal development. CONCLUSION: The 20E biosynthesis was regulated by the expression of CYP314A1, and was involved in the induction and termination phase of obligate diapause by regulating energy metabolism in B. minax. © 2022 Society of Chemical Industry.

How asymmetric DNA replication achieves symmetrical fidelity.

Accurate DNA replication of an undamaged template depends on polymerase selectivity for matched nucleotides, exonucleolytic proofreading of mismatches, and removal of remaining mismatches via DNA mismatch repair (MMR). DNA polymerases (Pols) δ and ε have 3'-5' exonucleases into which mismatches are partitioned for excision in cis (intrinsic proofreading). Here we provide strong evidence that Pol δ can extrinsically proofread mismatches made by itself and those made by Pol ε, independently of both Pol δ's polymerization activity and MMR. Extrinsic proofreading across the genome is remarkably efficient. We report, with unprecedented accuracy, in vivo contributions of nucleotide selectivity, proofreading, and MMR to the fidelity of DNA replication in Saccharomyces cerevisiae. We show that extrinsic proofreading by Pol δ improves and balances the fidelity of the two DNA strands. Together, we depict a comprehensive picture of how nucleotide selectivity, proofreading, and MMR cooperate to achieve high and symmetrical fidelity on the two strands.

LncRNA NEAT1 regulated diabetic retinal epithelial-mesenchymal transition through regulating miR-204/SOX4 axis.

AIM: Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells is the key of the development of diabetic retinopathy (DR), and lncRNA NEAT1 could accelerate EMT in diabetic nephropathy. Meanwhile, as a diabetes susceptibility gene, whether sex-determining region Y-related (SRY) high-mobility group box 4 (SOX4) has relationship with lncRNA NEAT1 in DR remains unclear. METHODS: Firstly, NEAT1, SOX4 and miR-204 were evaluated by qRT-PCR (quantitative reverse-transcriptase PCR) under high glucose condition. Then, cell viability, proliferation, migration and invasion were respectively detected by MTT, BrdU staining, wound healing and transwell assay after NEAT1 knockdown or miR-204 overexpression. Also, the EMT-related proteins were examined by western blot and cell immunofluorescence assay. In order to confirm the relationship between miR-204 and NEAT1 or SOX4, dual luciferase reporter gene assay was conducted. At the same time, the protein levels of SOX4 and EMT-related proteins were investigated by immunohistochemistry in vivo. RESULTS: High glucose upregulated NEAT1 and SOX4 and downregulated miR-204 in ARPE19 cells. NEAT1 knockdown or miR-204 overexpression inhibited the proliferation and EMT progression of ARPE19 cells induced by high glucose. NEAT1 was identified as a molecular sponge of miR-204 to increase the level of SOX4. The effect of NEAT1 knockdown on the progression of EMT under high glucose condition in ARPE19 cells could be reversed by miR-204 inhibitor. Also, NEAT1 knockdown inhibited retinal EMT in diabetic mice. CONCLUSION: NEAT1 regulated the development of EMT in DR through miR-204/SOX4 pathway, which could provide reference for clinical prevention and treatment.

Comparative transcriptome analysis of different developmental stage of Bactrocera minax (Diptera: Tephritidae): Implication of the molecular basis of its obligatory diapause induction.

The Chinese citrus fruit fly, Bactrocera minax (Enderlein), a devastating pest in citrus, has an obligatory diapause at the pupal stage. Although the physiology of obligatory diapause has been well described, the molecular mechanisms underlying the process remain unknow. Here we investigated the molecular mechanisms of obligatory diapause induction in B. minax using high-throughput RNA-Seq data from second-instar larva (2L), third-instar larva (3L) and pupa (P) stages. A total of 116,402 unigenes were obtained, of which 54,781 unigenes were successfully annotated in public databases, and the differentially expressed genes in the 3L vs 2L, P vs 2L, and P vs 3L comparisons were identified. The cluster co-expression patterns of the differentially expressed genes revealed that significantly differentially-expressed genes in the pupal stage were predicted to be related to diapause induction. All differentially expressed genes were investigated by GO functional and KEGG pathway analysis, and the results showed that genes involved in processes such as 20-hydroxyecdysone (20E) biosynthesis, cell cycle and metabolic pathways are likely related to obligatory diapause induction in B. minax. These results provide important information on the transcriptome of the Chinese citrus fruit fly that can be used for further functional studies as well as contributing to our understanding of the molecular basis of obligatory diapause induction and suggesting potential molecular targets for the control of this pest.

Comparison of canal transportation and centering ability of manual K-files and reciprocating files in glide path preparation: a micro-computed tomography study of constricted canals.

BACKGROUND: Optimum Glide Path (OGP) is a new reciprocating motion aiming to perform efficient glide path preparation in constricted canals. The aim of this study was to investigate and compare manual and OGP movement in terms of canal transportation and centering ability in glide path preparation of constricted canals. METHODS: Thirty constricted mesial root canals of mandibular molars, with initial apical size no larger than ISO#8, were selected and negotiated with #6-#8 K-files under the microscope. Canals were randomly divided into two experimental groups: Group 1 (MAN, n = 15): Glide path was established by using #10-#15 stainless steel K-files manually; Group 2 (OGP, n = 15): #10-#15 Mechanical Glide Path super-files were used with OGP motion (OGP 90°, 300 rpm). Each instrument was used to prepare only 2 canals (as in one mesial root). Canals were scanned before and after glide path preparation with micro-computed tomography (micro-CT) to evaluate root canal transportation and centering ratio at 1, 3 and 5 mm levels from the root apex. File distortions and separations were recorded. Paired t-test was used to statistically evaluate the data (P < .05). RESULTS: Group 2 showed a significantly lower transportation value than group 1 at 1-mm and 3-mm levels (P < .05), however the difference at 5-mm level was not significant. There was no significant difference regarding the centering ratio between the groups. Six #10 K-files were severely distorted in group 1, while no file separation or distortion was found in group 2. CONCLUSIONS: OGP motion performed significantly less canal transportation (apical 3 mm) and file distortion during glide path establishment in constricted canals comparing to manual motion, while the centering ability between the two was similar. CLINICAL RELEVANCE: OGP reciprocating motion provides a safer and efficient clinical approach compared to traditional manual motion in glide path establishment with small files in constricted canals.

Ribonucleotide incorporation into DNA during DNA replication and its consequences.

Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology. We also discuss ribonucleotide processing, the genetic consequences of unrepaired ribonucleotides in DNA and evidence supporting the significance of their transient presence in the nuclear genome.

DNA Polymerase Delta Synthesizes Both Strands during Break-Induced Replication.

Break-induced replication (BIR) is a pathway of homology-directed repair that repairs one-ended DNA breaks, such as those formed at broken replication forks or uncapped telomeres. In contrast to conventional S phase DNA synthesis, BIR proceeds by a migrating D-loop and results in conservative synthesis of the nascent strands. DNA polymerase delta (Pol δ) initiates BIR; however, it is not known whether synthesis of the invading strand switches to a different polymerase or how the complementary strand is synthesized. By using alleles of the replicative DNA polymerases that are permissive for ribonucleotide incorporation, thus generating a signature of their action in the genome that can be identified by hydrolytic end sequencing, we show that Pol δ replicates both the invading and the complementary strand during BIR. In support of this conclusion, we show that depletion of Pol δ from cells reduces BIR, whereas depletion of Pol ε has no effect.

Roles for DNA polymerase δ in initiating and terminating leading strand DNA replication.

Most current evidence indicates that DNA polymerases ε and δ, respectively, perform the bulk of leading and lagging strand replication of the eukaryotic nuclear genome. Given that ribonucleotide and mismatch incorporation rates by these replicases influence somatic and germline patterns of variation, it is important to understand the details and exceptions to this overall division of labor. Using an improved method to map where these replicases incorporate ribonucleotides during replication, here we present evidence that DNA polymerase δ universally participates in initiating leading strand synthesis and that nascent leading strand synthesis switches from Pol ε to Pol δ during replication termination. Ribonucleotide maps from both the budding and fission yeast reveal conservation of these processes. These observations of replisome dynamics provide important insight into the mechanisms of eukaryotic replication and genome maintenance.

Genome-wide mutagenesis resulting from topoisomerase 1-processing of unrepaired ribonucleotides in DNA.

Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2-5 base pair deletions (Δ2-5bp) in repetitive DNA are a signature of unrepaired ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol ε, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis.

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Evidence that DNA polymerase δ contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae.

To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). Although pol2-16 mutants survive, they present very tiny spore colonies, increased doubling time, larger than normal cells, aberrant nuclei, and rapid acquisition of suppressor mutations. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack only Pol ε proofreading (pol2-4), consistent with the idea that Pol ε is the major leading-strand polymerase used for unstressed DNA replication. Ribonucleotides are incorporated into the pol2-16 genome in patterns consistent with leading-strand replication by Pol δ when Pol ε is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.

Strand-specific analysis shows protein binding at replication forks and PCNA unloading from lagging strands when forks stall.

In eukaryotic cells, DNA replication proceeds with continuous synthesis of leading-strand DNA and discontinuous synthesis of lagging-strand DNA. Here we describe a method, eSPAN (enrichment and sequencing of protein-associated nascent DNA), which reveals the genome-wide association of proteins with leading and lagging strands of DNA replication forks. Using this approach in budding yeast, we confirm the strand specificities of DNA polymerases delta and epsilon and show that the PCNA clamp is enriched at lagging strands compared with leading-strand replication. Surprisingly, at stalled forks, PCNA is unloaded specifically from lagging strands. PCNA unloading depends on the Elg1-containing alternative RFC complex, ubiquitination of PCNA, and the checkpoint kinases Mec1 and Rad53. Cells deficient in PCNA unloading exhibit increased chromosome breaks. Our studies provide a tool for studying replication-related processes and reveal a mechanism whereby checkpoint kinases regulate strand-specific unloading of PCNA from stalled replication forks to maintain genome stability.

Fission yeast Pxd1 promotes proper DNA repair by activating Rad16XPF and inhibiting Dna2.

Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3' endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5' endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases.

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method.

Spontaneous DNA damage may occur nonrandomly in the genome, especially when genome maintenance mechanisms are undermined. We developed single-strand DNA (ssDNA)-associated protein immunoprecipitation followed by sequencing (SPI-seq) to map genomic hotspots of DNA damage. We demonstrated this method with Rad52, a homologous recombination repair protein, which binds to ssDNA formed at DNA lesions. SPI-seq faithfully detected, in fission yeast, Rad52 enrichment at artificially induced double-strand breaks (DSBs) as well as endogenously programmed DSBs for mating-type switching. Applying Rad52 SPI-seq to fission yeast mutants defective in DNA helicase Pfh1 or histone H3K56 deacetylase Hst4, led to global views of DNA lesion hotspots emerging in these mutants. We also found serendipitously that histone dosage aberration can activate retrotransposon Tf2 and cause the accumulation of a Tf2 cDNA species bound by Rad52. SPI-seq should be widely applicable for mapping sites of DNA damage and uncovering the causes of genome instability.