Replication Block Research Articles

Disruption of origin chromatin structure by helicase activation in the absence of DNA replication.

Prior to initiation of DNA replication, the eukaryotic helicase, Mcm2-7, must be activated to unwind DNA at replication start sites in early S phase. To study helicase activation within origin chromatin, we constructed a conditional mutant of the polymerase α subunit Cdc17 (or Pol1) to prevent priming and block replication. Recovery of these cells at permissive conditions resulted in the generation of unreplicated gaps at origins, likely due to helicase activation prior to replication initiation. We used micrococcal nuclease (MNase)-based chromatin occupancy profiling under restrictive conditions to study chromatin dynamics associated with helicase activation. Helicase activation in the absence of DNA replication resulted in the disruption and disorganization of chromatin, which extends up to 1 kb from early, efficient replication origins. The CMG holohelicase complex also moves the same distance out from the origin, producing single-stranded DNA that activates the intra-S-phase checkpoint. Loss of the checkpoint did not regulate the progression and stalling of the CMG complex but rather resulted in the disruption of chromatin at both early and late origins. Finally, we found that the local sequence context regulates helicase progression in the absence of DNA replication, suggesting that the helicase is intrinsically less processive when uncoupled from replication.

Determination of DNA lesion bypass using a ChIP-based assay

DNA lesion bypass facilitates DNA synthesis across bulky DNA lesions, playing a critical role in DNA damage tolerance and cell survival after DNA damage. Assessing lesion bypass efficiency in the cell is important to better understanding of the mechanism of carcinogenesis and chemoresistance. Here we developed a chromatin immunoprecipitation (ChIP)-based method to measure lesion bypass activity across cisplatin-induced intrastrand crosslinks in cancer cells. DNA lesion bypass enables the replication to continue in the presence of replication blocks. Thus, the successful lesion bypass should result in the coexistence of DNA lesions and the newly synthesized DNA fragment opposite to this lesion. Using ChIP, we precipitated the cisplatin-induced intrastrand crosslinks, and quantitated the precipitated newly synthesized DNA that was labeled with BrdU. We validated this method on ovarian cancer cells with inhibited TLS activity. We then applied this method to show that ovarian cancer stem cells exhibit high lesion bypass activity relative to bulk cancer cells from the same cell line. In conclusion, this novel ChIP-based lesion bypass assay can detect the extent to which cisplatin-induced DNA lesions are bypassed in live cells. Our study may be applied more broadly to the study of other DNA lesions, as specific antibodies to these specific lesions are available.

Rearrangement of incomplete multi-omics datasets combined with ComDim for evaluating replicate cross-platform variability and batch influence

Multi-omics studies can highlight the interrelationships among data across different layers of biological information. However, methods for the unsupervised analysis of multi-block data do not take the individual variability across batches into account and cannot deal with omics datasets when they present different numbers of replicates. We have explored three different data arrangement strategies to tackle these limitations. Several multi-block methods can be used to decipher the common variations across blocks and to determine the contribution of each block to each common component. In this study the ComDim method was used to compare these rearrangement strategies for three multi-omics datasets. We found that arranging the data using the ‘replicate by blocks’ strategy, where each block comprises data from only one replicate independently of its data type, provided the most insightful results. ComDim allowed the evaluation of the variability across the replicate blocks, confirming the existence of batch effects in some of the studies. Moreover, since the contributions of these batch effects were separated from the other contributions, the coordinated biological responses common across the different blocks was characterized for each data type.

Effects of organic fertilizer on growth and yield of tomato

The experiment was conducted during rabi season 2019-2020 at Agricultural Research Station, On Farm Research Division, Alamnagar, Rangpur to find out the useful effects of organic fertilizer on growth and yield of tomato. The experiment was arranged in a randomized complete block design (RCBD) with five treatments in three (03) compacted replicate blocks. The treatments included T1: 100% Recommended Chemical Fertilizer (RCF), T2: 85% CF + 3 tha-1 organic Fertilizer (OF), T3: 85% CF + 1 tha-1 OF, T4: 70% CF + 3 tha-1 OF and T5: 70% CF + 1 tha-1 OF. The highest yield was observed in T2 (50.59 t ha-1) due to more number of fruit plant-1& weight of fruit plant-1 and the lowest was in T5 (35.32 t ha-1). These results may be due the parameters of growth components increased with increasing amount of organic and inorganic fertilizers applied. Combination of organic and inorganic fertilizer treated plots produced higher yield than plots without combination of organic and inorganic fertilizer. The highest gross return (BDT. 607080) was found in T2 treatment and the lowest gross return (BDT. 423840) was recorded from T5. The highest gross margin (BDT. 328520 ha-1) was obtained from T2. The lowest gross margin (Tk. 145280 ha-1) was obtained from T5. Integrated nutrient management (combination of organic and inorganic fertilizer) is the best option for higher tomato production in Bangladesh.
 Progressive Agriculture 32 (1): 10-16, 2021

A wake-up call for cancer DNA damage: the role of Schlafen 11 (SLFN11) across multiple cancers.

DNA-damaging agents exploit increased genomic instability, a hallmark of cancer. Recently, inhibitors targeting the DNA damage response (DDR) pathways, such as PARP inhibitors, have also shown promising therapeutic potential. However, not all tumors respond well to these treatments, suggesting additional determinants of response are required. Schlafen 11 (SLFN11), a putative DNA/RNA helicase that induces irreversible replication block, is emerging as an important regulator of cellular response to DNA damage. Preclinical and emerging clinical trial data suggest that SLFN11 is a predictive biomarker of response to a wide range of therapeutics that cause DNA damage including platinum salts and topoisomerase I/II inhibitors, as well as PARP inhibitors, which has raised exciting possibilities for its clinical application. In this article, we review the function, prevalence, and clinical testing of SLFN11 in tumor biopsy samples and circulating tumor cells. We discuss mounting evidence of SLFN11 as a key predictive biomarker for a wide range of cancer therapeutics and as a prognostic marker across several cancer types. Furthermore, we discuss emerging areas of investigation such as epigenetic reactivation of SLFN11 and its role in activating immune response. We then provide perspectives on open questions and future directions in studying this important biomarker.

Genome-wide mapping reveals R-loops associated with centromeric repeats in maize.

R-loops are stable chromatin structures comprising a DNA:RNA hybrid and a displaced single-stranded DNA. R-loops have been implicated in gene expression and chromatin structure, as well as in replication blocks and genome instability. Here, we conducted a genome-wide identification of R-loops and identified more than 700,000 R-loop peaks in the maize (Zea mays) genome. We found that sense R-loops were mainly enriched in promoters and transcription termination sites and relatively less enriched in gene bodies, which is different from the main gene-body localization of sense R-loops in Arabidopsis and Oryza sativa. At the chromosome scale, maize R-loops were enriched in pericentromeric heterochromatin regions, and a significant portion of R-loops were derived from transposable elements. In centromeres, R-loops preferentially formed within the binding regions of the centromere-specific histone CENH3, and centromeric retrotransposons were strongly associated with R-loop formation. Furthermore, centromeric retrotransposon R-loops were observed by applying the single-molecule imaging technique of atomic force microscopy. These findings elucidate the fundamental character of R-loops in the maize genome and reveal the potential role of R-loops in centromeres.

Monsavirus in monkey rectal swab and throat swab specimens in China: Proposal for Posaliviridae as a new family in Picornavirales

Posa-like viruses have been detected in the fecal samples of several host species and are considered unclassified members of Picornavirales. Here, we identified genomic fragments of novel posa-like viruses (monsaviruses) in monkey specimens through next generation sequencing and obtained 11 full-length genomes. This monsavirus shared 88.5–89.2% nucleotide similarity with the Tottori-HG1 strain (GenBank accession LC123275). In total, 713 nucleotide polymorphism sites were identified, indicating their persistent evolution during circulation. The genomic organization and phylogenetic relationship of monsavirus were determined. Subsequent phylogenetic analysis of the conserved replication block of Hel-Pro-RdRp and core RNA-dependent RNA polymerase domain-based analysis of posa-like viruses showed significant separation compared with other known families. Further, posa-like virus genomes possessed the classical replication block of picornavirus in the 5′ part of genome and picorna-like capsid domains at the structural coding region of 3′ part of genome. Based on these results, we proposed the new family Posaliviridae, within Picornavirales. Four genera, which showed 68.6–75.5% amino acid distances but similar genomic organization including the conserved replication block of Hel-Pro-RdRp, the same order of the genomic coding region, and picorna-like capsid domains, were identified. The flexible genomic organization strategy and a large evolutionary scale of Posaliviridae was explicit. This study provides novel information on monsaviruses and important taxonomic data for the family Posaliviridae.

Abstract 1461: PHI-101, a potent and novel inhibitor of CHK2 in ovarian and breast cancer cells

Abstract Purpose: Development of the potent and selective checkpoint kinase 2 (CHK2) inhibitor to overcome limiting clinical utility of poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. Experimental Procedure: Preclinical evaluation of PHI-101 for cellular and molecular potency in ovarian and breast cancer cell lines and patient derived primary cells; it includes biochemical binding assay, cellular assays, animal efficacy studies, combination study, signaling pathway effect examination, and cell cycle analysis. Summary: CHK2 is a serine/threonine kinase and a cell cycle checkpoint regulator involved in the ATM-mediated DNA repair upon replication blocks and DNA damage. It has been proposed that Chk2 functions as a barrier to tumorigenesis by maintaining genomic stability, and this DNA damage induction is thought to prevent or delay genetic instability and tumorigenesis. The inhibition of CHK2 is a promising approach to achieve synthetic lethality of cancer cells when combined with PARP1 inhibitors. Solid cancer indications of PHI-101 was identified by the Chemiverse Network module which is an AI and Big data-based in-house drug discovery platform. Biochemical kinase assays for PHI-101 showed stronger affinity to CHK2 over CHK1 more than 5-fold. PHI-101 treatment of ovarian and breast cancer cells for 72 hrs elicit a synergistic lethal response in combination with PARP1 inhibitor Olaparib regardless of functional BRCA and P53 in the cells. PHI-101 also potentiates a countermeasure to dose-limiting toxicity triggered by genotoxic agents such as cisplatin and topotecan. The present results from in vivo and in vitro preclinical testing do demonstrate that PHI-101 is a highly potent inhibitor of CHK2 and may exert mono- and combinational therapeutic activity in ovarian and breast cancer model. Conclusion: The preclinical evaluation of PHI-101, a novel CHK2 inhibitor, showed clear evidence of anticancer activity for refractory ovarian and breast cancer cells and improved efficacy in both in vitro and in vivo models. Consequently, PHI-101 is currently under investigation in Phase 1 clinical trials for relapsed or refractory ovarian cancer patients. Citation Format: June H-J Han, Kyu-Tae Kim, Jeejin Im, Sojung Park, Min Kyung Choi, Inki Kim, Ky-Youb Nam, JeongHyeok Yoon. PHI-101, a potent and novel inhibitor of CHK2 in ovarian and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1461.

Abstract 1640: Correlations of DNA damage response gene alterations with response to immune checkpoint inhibitors are different in solid tumors

Abstract Background: The DNA damage response (DDR), which is frequently disrupted in cancer, is a signal transduction pathway that responds to DNA damage and DNA replication blocks. Genomic alterations in DDR genes are associated with increased genomic instability and higher tumor mutational burden (TMB). A better understanding of DDR alterations will not only expand our knowledge of cancer development but also led to new cancer treatments. TMB has different predictive values among multiple cancer types. We hypothesized that the prediction values of DDR alterations for response to immune checkpoint inhibitors (ICIs) are also varied. In the present work, we explored the associations between DDR alterations and ICIs efficacy in different solid tumors. Methods: In a cohort of 3396 patients at our center were enrolled. The following tumor types are included: NSCLC (n=1080), colorectal cancer (n=509), gastric cancer (n=269), bile duct cancer (n=122), pancreatic cancer (n=199), hepatocellular carcinoma (n=309), glioma (n=778), melanoma (130). Targeted next-generation sequencing and PD-L1 immunohistochemistry (22C3 antibody) were performed. The ICIs treatment cohort from the Memorial Sloan Kettering Cancer Center (MSKCC) was also selected. Results: Across all 3396 patients, the mutational frequencies of DDR genes were 23.4%. Hepatocellular carcinoma, colorectal cancer, and NSCLC were the tumor types in which DDR alterations were most frequent-30.4%, 30.1% , and 29.1%, respectively. About 7.6% of patients have high microsatellite instability (MSI-H) in the DDR alterations group, while 0.2% of patients display MSI-H in the control group (P < 0.0001). For all patients, the median TMB was significantly higher in the DDR alterations group (8.92 vs 4.69 muts/Mb, P < 0.0001). Furthermore, 39.7% of patients had PD-L1 expression (TPS≥10%) in the DDR alterations group and 29.9% of patients had PD-L1 expression in the control group (P = 0.016). For the entire MSKCC cohort, patients in the DDR alterations group had significantly longer median overall survival after ICIs therapy (34 vs 15 months, P < 0.0001). However, DDR alterations display divergent predictive roles in different cancer types. DDR alterations are associated with ICIs efficacy in melanoma (P = 0.0007), colorectal cancer (P = 0.0012), NSCLC (P = 0.0224), bladder cancer (P = 0.0439), and breast cancer (P = 0.0451). There were no associations between DDR alterations and benefit from ICIs in renal cell carcinoma, glioma, esophagogastric, and head and neck cancers. Conclusions: Our data indicated that DDR alterations are associated with increased TMB, MSI-H, and higher PD-L1 expression. Furthermore, DDR alterations were associated with better response to ICIs in pan-cancer analysis. It's worth noting that DDR alterations can predict patient responses to ICIs in some cancer types, but not in all types of cancer. Citation Format: Tao Li, Yanling Niu, Tonghui Ma, Danhua Wang, Dong-Dong Jia. Correlations of DNA damage response gene alterations with response to immune checkpoint inhibitors are different in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1640.

Seedling Growth Analysis of Papaya Cultivated on Several Planting Media Enriched by Plant Growth Promotor Microbes

<p class="KataPengantar">There are factors contributed to the growth and development of fruit crop seedling. Microbes are well known as plant growth promotors such as symbiotic mycorrhizae and antagonist fungi, <em>Trichoderma </em>spp. The main objective of this experiment is to find out the best medium composition enriched by beneficial microbes to improve papaya seedling growth. The experiment was conducted at Sumani Experimental Station, Indonesian Tropical Fruit Research Institute, Solok, West Sumatera, Indonesia from August until December 2017. The experiment was arranged in a Randomized Complete Block Design with ten treatments and three replicate blocks. The treatments were ten combinations of media for papaya seedling growth enriched by plant growth promotor microbes. In this experiment, medium soil with additional manure, rice husk charcoal and compost (single or combination) combined with mycorrhizae were used. Also, the effect of <em>Trichoderma </em><em>sp</em><em>.</em> enrichment into media composition were tested in this experiment. Treatments SCRMc and SCRMcT; with its complexity; were the best media composition to promote papaya seedling growth. These treatments resulted in best performance of plant height, stem diameter and number of leaves of papaya seedlings. The additional of <em>Trichoderma </em><em>sp</em><em>.</em> into medium did not show beneficial effect for all parameters in this experiment.</p>

Effects of pre-, pro-, and synbiotic supplementation on the growth performance and feed conversion rates of Indonesian native chicken – The offspring of in ovo L-gln fed hen

A research was conducted to elucidate the effect of probiotic, prebiotic and synbiotic supplementation in drinking water on the performance of Indonesian Native Chickens (INC), the offspring of in ovo 1.5% L-gln hens. A randomized Block Design of 4 treatments (P0=control; P1=Prebiotic (10 g Inulin in 1 l tap water); P2= Probiotic (2 g Promix in 1 l tap water), and P3=Synbiotic (10 g Inulin + 2 g Promix in 1 l tap water) with 3 times of replication blocks was used. An amount of 180 one-day-old (DOC) of INC from 3 hatching periods was used in the research, which was selected from newly hatched chicks as the offsprings of laying hen resulted from the in ovo L-gln. The chicks were put in brooding boxes separately for each treatment unit (maximum 15 chicks per box) and placed in a rearing room (27-280C; 60% RH). The results indicated that supplementation of pre-, pro-, and symbiotic during the first 2 weeks through drinking water did not significantly affect the body weight and feed conversion ratio (FCR) of INC; Afterward, measuring at weeks 4, 6, and 8, supplementation resulted in significantly heavier body weights and better FCR compared to those of control chicks. In conclusion, this study has shown that supplementation of pre-, pro-, and symbiotic exert positive effects on the growth and FCR of INC, and the best response is to symbiotic supplementation.

Structural understanding of non-nucleoside inhibition in an elongating herpesvirus polymerase

All herpesviruses encode a conserved DNA polymerase that is required for viral genome replication and serves as an important therapeutic target. Currently available herpesvirus therapies include nucleoside and non-nucleoside inhibitors (NNI) that target the DNA-bound state of herpesvirus polymerase and block replication. Here we report the ternary complex crystal structure of Herpes Simplex Virus 1 DNA polymerase bound to DNA and a 4-oxo-dihydroquinoline NNI, PNU-183792 (PNU), at 3.5 Å resolution. PNU bound at the polymerase active site, displacing the template strand and inducing a conformational shift of the fingers domain into an open state. These results demonstrate that PNU inhibits replication by blocking association of dNTP and stalling the enzyme in a catalytically incompetent conformation, ultimately acting as a nucleotide competing inhibitor (NCI). Sequence conservation of the NCI binding pocket further explains broad-spectrum activity while a direct interaction between PNU and residue V823 rationalizes why mutations at this position result in loss of inhibition.

Multinucleation associated DNA damage blocks proliferation in p53-compromised cells

Nuclear atypia is one of the hallmarks of cancers. Here, we perform single-cell tracking studies to determine the immediate and long-term impact of nuclear atypia. Tracking the fate of newborn cells exhibiting nuclear atypia shows that multinucleation, unlike other forms of nuclear atypia, blocks proliferation in p53-compromised cells. Because ~50% of cancers display compromised p53, we explored how multinucleation blocks proliferation. Multinucleation increases 53BP1-decorated nuclear bodies (DNA damage repair platforms), along with a heterogeneous reduction in transcription and protein accumulation across the multi-nucleated compartments. Multinucleation Associated DNA Damage associated with 53BP1-bodies remains unresolved for days, despite an intact NHEJ machinery that repairs laser-induced DNA damage within minutes. Persistent DNA damage, a DNA replication block, and reduced phospho-Rb, reveal a novel replication stress independent cell cycle arrest caused by mitotic lesions. These findings call for segregating protective and prohibitive nuclear atypia to inform therapeutic approaches aimed at limiting tumour heterogeneity.

Individual and Additive Effects of Insecticide and Mating Disruption in Integrated Management of Navel Orangeworm in Almonds.

Simple SummaryMating disruption is an increasingly important part of pest management for the navel orangeworm Amyelois transitella. Industry groups have long supported mating disruption research and development with the divergent objectives of both minimizing damage from this key pest and reducing insecticide used on these crops. It is therefore important to know whether the benefits of mating disruption and insecticide are additive or, alternatively, if using both together provides no additional benefit over either alone. Ten years of data from research trials in a large commercial almond orchard found that the benefits of mating disruption are generally additive with lower damage if both are used together than either alone. Substantial year-to-year variability in navel orangeworm damage was also evident, even with stringent management. These findings indicate that the combination of mating disruption and insecticide can reduce the impact of navel orangeworm damage on the almond industry. Further improvements in monitoring and predictions of navel orangeworm abundance and damage are necessary for mating disruption to effectively contribute to the industry goal of reduction of insecticide use by 25%.Damage from Amyelois transitella, a key pest of almonds in California, is managed by destruction of overwintering hosts, timely harvest, and insecticides. Mating disruption has been an increasingly frequent addition to these management tools. Efficacy of mating disruption for control of navel orangeworm damage has been demonstrated in experiments that included control plots not treated with either mating disruption or insecticide. However, the navel orangeworm flies much farther than many orchard pests, so large plots of an expensive crop are required for such research. A large almond orchard was subdivided into replicate blocks of 96 to 224 ha and used to compare harvest damage from navel orangeworm in almonds treated with both mating disruption and insecticide, or with either alone. Regression of navel orangeworm damage in researcher-collected harvest samples from the interior and center of management blocks on damage in huller samples found good correlation for both and supported previous assumptions that huller samples underreport navel orangeworm damage. Blocks treated with both mating disruption and insecticide had lower damage than those treated with either alone in 9 of the 10 years examined. Use of insecticide had a stronger impact than doubling the dispenser rate from 2.5 to 5 per ha, and long-term comparisons of relative navel orangeworm damage to earlier- and later-harvested varieties revealed greater variation than previously demonstrated. These findings are an economically important confirmation of trade-offs in economic management of this critical pest. Additional monitoring tools and research tactics will be necessary to fulfill the potential of mating disruption to reduce insecticide use for navel orangeworm.

Transcriptome of Cydia pomonella granulovirus in susceptible and type I resistant codling moth larvae.

The baculovirus Cydia pomonella granulovirus (CpGV) is a biocontrol agent used worldwide against the codling moth (CM), Cydia pomonella L., a severe pest in organic and integrated pome fruit production. Its successful application is increasingly challenged by the occurrence of CM populations resistant to commercial CpGV products. Whereas three types (I–III) of CpGV resistance have been identified, type I resistance compromising the efficacy of CpGV-M, the so-called Mexican isolate of CpGV, is assumed to be the most widely distributed resistance type in Central Europe. Despite the wide use of CpGV products as biocontrol agents, little information is available on gene-expression levels in CM larvae. In this study, the in vivo transcriptome of CpGV-M infecting susceptible (CpS) and resistant (CpRR1) CM larvae was analysed at 24, 48, 72, 96 and 120 hours post infection in the midgut and fat body tissue by using a newly developed microarray covering all ORFs of the CpGV genome. According to their transcript abundance, the CpGV genes were grouped into four temporal clusters to which groups of known and unknown function could be assigned. In addition, sets of genes differentially expressed in the midgut and fat body were found in infected susceptible CpS larvae. For the resistant CpRR1 larvae treated with CpGV-M, viral entry in midgut cells could be confirmed from onset but a significantly reduced gene expression, indicating that type I resistance is associated with a block of viral gene transcription and replication.

Effects of broiler genetic strain and dietary amino acid reduction on meat yield and quality (part II)

Genetic selection and advances in nutrition have improved broiler growth performance. However, meat quality issues have gained preference over increased growth rate. These meat quality issues may be reduced by lowering dietary amino acid (AA) content. In the present study, 5 common commercial broiler strains were fed either a control or an AA-reduced diet. The control diet was formulated to contain the highest digestible AA (lysine, total sulfur AA, and threonine) levels recommended for the 5 strains. The AA-reduced diet was formulated to contain 20% lower levels of these 3 digestible AA than in the control diet. This resulted in a 5 (strains) × 2 (AA levels) factorial arrangement. A total of 1,280 straight run broilers were randomly allocated to 8 replicate blocks. The AA reduction decreased absolute breast weights of 3 strains on day 42 and 2 strains on day 56, and decreased absolute weights of tender, wing, drumstick, and thigh on both day 42 and 56 for all 5 strains. However, the absolute fat pad weight and relative fat pad and thigh weights to BW were increased in the AA reduction treatments on both day 42 and 56. The AA reduction contributed to the lower breast meat pH on both day 42 and 56, which may have been directly related to decreased severe woody breast myopathy (WBM) incidence on day 42 and moderate WBM incidence on day 56. The severity of WBM was positively related to breast weight in all 10 treatments on both day 42 and 56, with the exception of birds in strain 3 on day 56 that were fed the AA-reduced diet. At the same time, AA reduction was more cost-effective when WBM incidence was considered in a theoretical model. In conclusion, WBM severity was associated with higher breast weight in birds of most strains fed either a control or AA-reduced diet. Dietary AA reduction decreased processing yields but decreased WBM incidence, which may be more economical.

Single-Molecule Dynamics at a Bacterial Replication Fork after Nutritional Downshift or Chemically Induced Block in Replication.

Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single-molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and lagging-strand polymerase DnaE in the model bacterium Bacillus subtilis, we show that proteins react differently to stress conditions in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after the arrest of polymerase, but binds less frequently after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone (p)ppGpp due to stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behavior of DnaG and DnaE suggests that both proteins are recruited independently to the forks rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension.IMPORTANCE All cells need to adjust DNA replication, which is achieved by a well-orchestrated multiprotein complex, in response to changes in physiological and environmental conditions. For replication forks, it is extremely challenging to meet with conditions where amino acids are rapidly depleted from cells, called the stringent response, to deal with the inhibition of one of the centrally involved proteins or with DNA modifications that arrest the progression of forks. By tracking helicase (DnaC), primase (DnaG), and polymerase (DnaE), central proteins of Bacillus subtilis replication forks, at a single molecule level in real time, we found that interactions of the three proteins with replication forks change in different manners under different stress conditions, revealing an intriguing plasticity of replication forks in dealing with replication obstacles. We have devised a new tool to determine rates of exchange between static movement (binding to a much larger complex) and free diffusion, showing that during stringent response, all proteins have highly increased exchange rates, slowing down overall replication, while inactivation of polymerase or replication roadblocks leaves forks largely intact, allowing rapid restart once obstacles are removed.

Global Genome Demethylation Causes Transcription-Associated DNA Double Strand Breaks in HPV-Associated Head and Neck Cancer Cells

Simple SummaryHigh levels of global genome methylation in HPV-associated head and neck cancer prompted us to explore demethylation as a potential treatment by determining mechanisms of its toxicity in HPV-positive head and neck cancer cells. Previously, we reported that demethylating drug 5-azaC stabilizes p53 and reduces the expression of HPV genes and matrix metalloproteinases in HPV+ head and neck cancer cells and tumors from patients enrolled in a 5-azaC window clinical trial. Here, we extended our understanding of toxicity caused by global demethylation in HPV-associated head and neck cancer cells by finding that 5-azaC treatment results in formation of DNA double strand breaks that depend on transcription and replication.High levels of DNA methylation at CpG loci are associated with transcriptional repression of tumor suppressor genes and dysregulation of DNA repair genes. Human papilloma virus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) have high levels of DNA methylation and methylation has been associated with dampening of an innate immune response in virally infected cells. We have been exploring demethylation as a potential treatment in HPV+ HNSCC and recently reported results of a window clinical trial showing that HNSCCs are particularly sensitive to demethylating agent 5-azacytidine (5-aza). Mechanistically, sensitivity is partially due to downregulation of HPV genes expression and restoration of tumor suppressors p53 and Rb. Here, for the first time, we show that 5-azaC treatment of HPV+ HNSCC induces replication and transcription-associated DNA double strand breaks (DSBs) that occur preferentially at demethylated genomic DNA. Blocking replication or transcription prevented formation of DNA DSBs and reduced sensitivity of HPV-positive head and neck cancer cells to 5-azaC, demonstrating that both replication and active transcription are required for formation of DSBs associated with 5-azaC.

Effect of Feeding Quails with Mixture Feeds Composed of Crab Waste Meal, Leubim Fish Waste Meal, and Broken Rice Grains as Partly Substitution of Commercial Diet on Egg Quality

In rearing quails, many attempts have been done to reduce feed cost among other things by replacing partly commercial diet with numerous alternative feed sources such as crab waste meal (CWM), leubim fish (Canthidermis maculata) waste meal (LFWM), and broken rice grains (BRG). The purpose of this study was to determine the effect of using a mixture feeds composed of CWM + LFWM + BRG as a substitute for commercial laying chicken diets on the quality of quail eggs. This research was conducted at the Field Laboratory of Animal Husbandry and the Laboratory of Poultry Production Science, Syiah Kuala University. This study used 80 female quails (Coturnix-coturnix japonica) females aged 4 weeks. The treatment was feeding quails with the commercial diet of laying hen (324-1M) of which 0, 10, 20, and 30% of the diets was substituted by the mixture feeds composed of CWM + LFWM + BRG. The study was performed into block randomized design (BRD) consisting of 4 treatments and 4 replicate blocks. The blocks were established based on the different initial body weights of 4-week ages of female quails. Each treatment was an experimental unit consisting of 5 female quails each. The measured parameters were Yolk Index (YI), yolk color, Albumen Index (AI), eggshell thickness, and egg weight. The results of the study indicated that using up to 30% mixture feeds composed of 7,5% CWM + 9,1% LFWM + 13,4% BRG as a substitute for commercial laying chicken diets most significantly increased yolk index and yolk color of quail eggs. However, the albumen index, eggshell thickness, and egg weight were not significantly affected.

ACCURACY OF GENETIC PARAMETERS ESTIMATION AND PREDICTION OF GENOTYPIC VALUES IN EUCALYPTUS USING DIFFERENT PLOT TYPES

A suitable experimental design should control the environmental effect and be capable of discriminating genotypes. Two clonal tests of Eucalyptus were planted side by side in a randomized block design with the same 32 treatments under two different plot designs. The first trial was designed with five plants per plot (linear plot, LP) and six block replicates at a spacing of 3 x 2 m. The second trial was designed with one plant per plot (single-tree plot, STP) and 30 block replicates also at a spacing of 3 x 2 m. Diameter at breast height (dbh), height (h), and individual volume (vol) were measured at five years of age. The STP design presented broad-sense heritabilities values of 0.23 for h and 0.41 for vol, which were higher than those found for the LP design (0.12 for h and 0.22 for vol). The ratio between the standard error and the estimated genotypic variance (Sg^ ) was lower for the three evaluated traits in the STP trial. The correlations between the genetic values were positive and of high magnitude, ranging from 0.87 (h) to 0.91 (vol), but there were many differences between the two designs with regard to the ranking of clones. The STP presented better discriminant power according to the biplot analysis, as well as more accurate and reliable estimates.