Chromosomal DNA Replication Research Articles

Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites.

Imbalance in the level of the pyrimidine degradation products dihydrouracil and dihydrothymine is associated with cellular transformation and cancer progression. Dihydropyrimidines are degraded by dihydropyrimidinase (DHP), a zinc metalloenzyme that is upregulated in solid tumors but not in the corresponding normal tissues. How dihydropyrimidine metabolites affect cellular phenotypes remains elusive. Here we show that the accumulation of dihydropyrimidines induces the formation of DNA–protein crosslinks (DPCs) and causes DNA replication and transcriptional stress. We used Xenopus egg extracts to recapitulate DNA replication invitro. We found that dihydropyrimidines interfere directly with the replication of both plasmid and chromosomal DNA. Furthermore, we show that the plant flavonoid dihydromyricetin inhibits human DHP activity. Cellular exposure to dihydromyricetin triggered DPCs-dependent DNA replication stress in cancer cells. This study defines dihydropyrimidines as potentially cytotoxic metabolites that may offer an opportunity for therapeutic-targeting of DHP activity in solid tumors.

Replication fidelity in E. coli: Differential leading and lagging strand effects for dnaE antimutator alleles

DNA Pol III holoenzyme (HE) is the major DNA replicase of Escherichia coli. It is a highly accurate enzyme responsible for simultaneously replicating the leading- and lagging DNA strands. Interestingly, the fidelity of replication for the two DNA strands is unequal, with a higher accuracy for lagging-strand replication. We have previously proposed this higher lagging-strand fidelity results from the more dissociative character of the lagging-strand polymerase. In support of this hypothesis, an E. coli mutant carrying a catalytic DNA polymerase subunit (DnaE915) characterized by decreased processivity yielded an antimutator phenotype (higher fidelity). The present work was undertaken to gain deeper insight into the factors that influence the fidelity of chromosomal DNA replication in E. coli. We used three different dnaE alleles (dnaE915, dnaE911, and dnaE941) that had previously been isolated as antimutators. We confirmed that each of the three dnaE alleles produced significant antimutator effects, but in addition showed that these antimutator effects proved largest for the normally less accurate leading strand. Additionally, in the presence of error-prone DNA polymerases, each of the three dnaE antimutator strains turned into mutators. The combined observations are fully supportive of our model in which the dissociative character of the DNA polymerase is an important determinant of in vivo replication fidelity. In this model, increased dissociation from terminal mismatches (i.e., potential mutations) leads to removal of the mismatches (antimutator effect), but in the presence of error-prone (or translesion) DNA polymerases the abandoned terminal mismatches become targets for error-prone extension (mutator effect). We also propose that these dnaE alleles are promising tools for studying polymerase exchanges at the replication fork.

Identification of survival-related predictors in hepatocellular carcinoma through integrated genomic, transcriptomic, and proteomic analyses

Patient survival time generally reflects the tumor progression and represents a key clinical parameter. In this study, we aimed to comprehensively characterize the prognosis-associated molecular alterations in hepatocellular carcinoma (HCC). In this study, copy-number changes, gene mutations, mRNA expression, and reverse phase protein arrays data in HCC samples profiled by The Cancer Genome Atlas (TCGA) were obtained. Tumors were then stratified into two groups based on the clinical outcome and identified genomic, transcriptomic, and proteomic traits associated to HCC prognosis. We found that several copy number amplifications and deletions can discriminate HCC patients with poor prognosis from those with better prognosis. Mutated DNAH8 showed a worse prognosis-specific pattern and correlated with a reduced disease-free survival in HCC. By integrating RNA sequencing data, we found that HCC samples with poor prognosis are consistently associated with the up-regulation of cell cycle process, such as chromosome separation, DNA replication, cytokinesis, and etc. At the proteomic level, seven proteins were significantly enriched in samples with poor prognosis, including acetylated α-Tubulin, p62-LCK-ligand, ARID1 A, MSH6, B-Raf, Cyclin B1, and PEA15. Acetylated α-Tubulin was frequently expressed in HCC tissues and acted as a promising prognostic factor for HCC. These alterations lay a foundation for developing relevant therapeutic strategies and improve our knowledge of the pathogenesis of HCC.

A new role for Escherichia coli Dam DNA methylase in prevention of aberrant chromosomal replication.

The Dam DNA methylase of Escherichia coli is required for methyl-directed mismatch repair, regulation of chromosomal DNA replication initiation from oriC (which is DnaA-dependent), and regulation of gene expression. Here, we show that Dam suppresses aberrant oriC-independent chromosomal replication (also called constitutive stable DNA replication, or cSDR). Dam deficiency conferred cSDR and, in presence of additional mutations (Δtus, rpoB*35) that facilitate retrograde replication fork progression, rescued the lethality of ΔdnaA mutants. The DinG helicase was required for rescue of ΔdnaA inviability during cSDR. Viability of ΔdnaA dam derivatives was dependent on the mismatch repair proteins, since such viability was lost upon introduction of deletions in mutS, mutH or mutL; thus generation of double strand ends (DSEs) by MutHLS action appears to be required for cSDR in the dam mutant. On the other hand, another DSE-generating agent phleomycin was unable to rescue ΔdnaA lethality in dam+ derivatives (mutS+ or ΔmutS), but it could do so in the dam ΔmutS strain. These results point to a second role for Dam deficiency in cSDR. We propose that in Dam-deficient strains, there is an increased likelihood of reverse replication restart (towards oriC) following recombinational repair of DSEs on the chromosome.

Crystal structure of the C-terminal domain of the primosomal DnaT protein: Insights into a new oligomerization mechanism

DnaT is a replication restart primosomal protein required for re-initiating chromosomal DNA replication in bacteria. DnaT can be a monomer, dimer, trimer, tetramer, or pentamer. The oligomerization and disassembly of DnaT oligomers are critical in primosome assembly. Prior to this work, only the ssDNA-bound structure of the pentameric DnaT truncated protein (aa 84–153; DnaT84–153) was available. The mechanism by which DnaT oligomerizes as different states is unclear. In this paper, we report the crystal structure of the C-terminal domain of DnaT (aa 84–179; DnaTc) at 2.30 Å resolution (PDB entry 6AEQ). DnaTc forms a dimer both in the crystalline state and in solution. As compared with the ssDNA-bound structure of the pentameric DnaT84–153, their subunit–subunit interfaces significantly differ. The different oligomeric architecture suggests a strong conformational change possibly induced by ssDNA. Superposition analysis further indicated that the monomer of a DnaTc dimer shifted away by a distance of 7.5 Å and rotated by an angle of 170° for binding to ssDNA. Basing from these molecular evidence, we discussed and proposed a working model to explain how DnaTc oligomerizes through residue R146 mediation.

Identification and characterization of stem-bulge RNAs in Drosophila melanogaster

ABSTRACTNon-coding Y RNAs and stem-bulge RNAs are homologous small RNAs in vertebrates and nematodes, respectively. They share a conserved function in the replication of chromosomal DNA in these two groups of organisms. However, functional homologues have not been found in insects, despite their common early evolutionary history. Here, we describe the identification and functional characterization of two sbRNAs in Drosophila melanogaster, termed Dm1 and Dm2. The genes coding for these two RNAs were identified by a computational search in the genome of D. melanogaster for conserved sequence motifs present in nematode sbRNAs. The predicted secondary structures of Dm1 and Dm2 partially resemble nematode sbRNAs and show stability in molecular dynamics simulations. Both RNAs are phylogenetically closer related to nematode sbRNAs than to vertebrate Y RNAs. Dm1, but not Dm2 sbRNA is abundantly expressed in D. melanogaster S2 cells and adult flies. Only Dm1, but not Dm2 sbRNA can functionally replace Y RNAs in a human cell-free DNA replication initiation system. Therefore, Dm1 is the first functional sbRNA described in insects, allowing future investigations into the physiological roles of sbRNAs in the genetically tractable model organism D. melanogaster.

Dynamic Exchange of Two Essential DNA Polymerases during Replication and after Fork Arrest

The replisome is a multiprotein machine responsible for the faithful replication of chromosomal and plasmid DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in live Bacillus subtilis cells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following replication fork stress using damage-independent and damage-dependent conditions. With these results, we report the dynamic and cooperative process of DNA replication based on changes in the measured diffusion coefficients and dwell times. These experiments show that the replication proteins are all highly dynamic and that the exchange rate depends on whether DNA synthesis is active or arrested. Our results also suggest coupling between PolC and DnaX in the DNA replication process and indicate that DnaX provides an important role in synthesis during repair. Furthermore, our results suggest that DnaE provides a limited contribution to chromosomal replication and repair in vivo.

Replisome Dynamics and Their Functional Relevance upon DNA Damage through the PCNA Interactome

SUMMARYEukaryotic cells use copious measures to ensure accurate duplication of the genome. Various genotoxic agents pose threats to the ongoing replication fork that, if not efficiently dealt with, can result in replication fork collapse. It is unknown how replication fork is precisely controlled and regulated under different conditions. Here, we examined the complexity of replication fork composition upon DNA damage by using a PCNA-based proteomic screen to uncover known and unexplored players involved in replication and replication stress response. We used camptothecin or UV radiation, which lead to fork-blocking lesions, to establish a comprehensive proteomics map of the replisome under such replication stress conditions. We identified and examined two potential candidate proteins WIZ and SALL1 for their roles in DNA replication and replication stress response. In addition, our unbiased screen uncovered many prospective candidate proteins that help fill the knowledge gap in understanding chromosomal DNA replication and DNA repair.

DNA Sliding Clamps as Therapeutic Targets.

Chromosomal DNA replication is achieved by an assembly of multi-protein complexes at the replication fork. DNA sliding clamps play an important role in this assembly and are essential for cell viability. Inhibitors of bacterial (β-clamp) and eukaryal DNA clamps, proliferating cell nuclear antigen (PCNA), have been explored for use as antibacterial and anti-cancer drugs, respectively. Inhibitors for bacterial β-clamps include modified peptides, small molecule inhibitors, natural products, and modified non-steroidal anti-inflammatory drugs. Targeting eukaryotic PCNA sliding clamp in its role in replication can be complicated by undesired effects on healthy cells. Some success has been seen in the design of peptide inhibitors, however, other research has focused on targeting PCNA molecules that are modified in diseased states. These inhibitors that are targeted to PCNA involved in DNA repair can sensitize cancer cells to existing anti-cancer therapeutics, and a DNA aptamer has also been shown to inhibit PCNA. In this review, studies in the use of both bacterial and eukaryotic sliding clamps as therapeutic targets are summarized.

Loss of RNF40 Decreases NF-κB Activity in Colorectal Cancer Cells and Reduces Colitis Burden in Mice.

Inflammatory bowel diseases are linked to an increased risk of developing colorectal cancer [CRC]. Previous studies suggested that the H2B ubiquitin ligase RING finger protein-20 [RNF20] inhibited inflammatory signaling mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB]. However, the role of RNF40, the obligate heterodimeric partner of RNF20, in the context of inflammation and CRC has not been addressed. Here, we examined the effect of RNF40 loss on CRC cells in vitro and on inflammation and inflammatory signaling in vitro and in vivo. We evaluated H2Bub1 levels in human and murine colorectal tumors by immunohistochemistry. Moreover, we correlated H2Bub1 and RNF40 levels in vivo and assessed the consequences of RNF40 depletion on cellular phenotype and gene expression in CRC cells in vitro. Finally, we examined the effect of a colon-specific loss of Rnf40 in a murine model of colitis, and assessed both local and systemic inflammation-associated consequences. In vitro studies revealed that the tumorigenic phenotype of CRC cells decreased after RNF40 depletion and displayed gene expression changes related to chromosome segregation and DNA replication, as well as decreased induction of several NF-κB-associated cytokines. This effect was associated with decreased nuclear localization of NF-κB following tumor necrosis factor alpha treatment. Consistently, the colon-specific loss of Rnf40 exerted a protective local, as well as systemic, effect following acute colitis. Our findings suggest that RNF40 plays a central role in the maintenance of tumorigenic features and inflammatory signaling by promoting nuclear NF-κB activity.

Identification of Survival-Related Predictors in Hepatocellular Carcinoma Through Integrated Genomic, Transcriptomic, and Proteomic Analyses

Patient survival time generally reflects the tumor progression and represents a key clinical parameter. In this study, using copy-number changes, gene mutations, mRNA expression, and reverse phase protein arrays data in hepatocellular carcinoma (HCC) samples with poor and better clinical outcome profiled by The Cancer Genome Atlas (TCGA), we performed comprehensive molecular characterization of prognosisassociated alterations. Among them, several copy number amplifications and deletions can discriminate HCC patients with poor prognosis from those with better prognosis. Mutated DNAH8 showed a worse prognosisspecific pattern and correlated with a reduced disease-free survival in HCC. By integrating RNA sequencing data, we found that HCC samples with poor prognosis are consistently associated with the up-regulation of cell cycle process, such as chromosome separation, DNA replication, cytokinesis, and etc. At the proteomic level, seven proteins were significantly enriched in samples with poor prognosis, including acetylated α-Tubulin, p62-LCK-ligand, ARID1A, MSH6, B-Raf, Cyclin B1, and PEA15. Acetylated α-Tubulin was frequently expressed in HCC tissues and acted as a promising prognostic factor for HCC. Collectively, these alterations lay a foundation for developing relevant therapeutic strategies and improve our knowledge of the pathogenesis of HCC. Funding Statement: The research was supported by grants from Shanghai Municipal Commission of Health and Family Planning, Key Developing Disciplines Program (No: 2015ZB0501), Shanghai Key disciplines program of Health and Family Planning (No: 2017ZZ02010), Shanghai Sailing Program (No: 17YF1405200), and National Natural Science Foundation of China (81701374). Declaration of Interests: The authors state: Nothing to report. Ethics Approval Statement: All human samples were obtained with informed consent, and protocols were approved by the ethical review committee of the World Health Organization Collaborating Center for Research in Human Production (authorized by the Shanghai Municipal Government).

Abstract 3292: Revisiting TCGA data identifies key genes in lung adenocarcinoma

Abstract Although the incidence rate of lung cancer has declined over the past few years, it remains the leading cause of cancer death worldwide. The Cancer Genome Atlas has provided invaluable resources for cancer research. The development of next generation sequencing technology and evolution of data analysis tools make it possible to interpret the underlying mechanism of oncogenesis. In current study, normalized FPKM data from 572 samples derived from patients with lung adenocarcinoma (LUAD) from TCGA was used to do weighted gene co-expression network analysis (WGCNA) to identify gene modules associated with lung adenocarcinoma. Raw counts data was used to identify differentially expressed genes in LUAD by using DESeq2 in R (version 3.4.2). WGCNA results shows that magenta (r = 0.47, p <0.001)), black (r = 0.40, p <0.001)) and yellow (r = -0.88, p <0.001) modules are top 3 modules that associated with LUAD and cancer stage. Gene Ontology over-representation analysis shows that genes in magenta modules are involved with transcript elongation; genes in black modules are enriched in cell meiosis related biological processes, including chromosome segregation, nuclear division, DNA replication and mitotic nuclear division et al; genes in yellow module are enriched in angiogenesis, leukocyte migration and hemostasis. It is noteworthy that genes in magenta and black modules are upregulated and in yellow modules are downregulated. GOLM1 and MUC4 in magenta module play certain roles in cancer development. Several genes in MEGA family, including MAGEA3, MAGEA6, MEGEA16 and MEGEA1, and IGF2BP1 in black module have been reported to be overexpressed in various kinds of tumors. However, whether these in silicon discovered genes can be used to distinguish LUAD from controls still needs to be further evaluated. Keywords: lung adenocarcinoma; TCGA; gene expression analysis; bioinformatics; cancer biomarker Citation Format: Jianxiang Shi, Mengtao Xing, Chenglin Luo, Xiao Wang, Liping Dai, Jianying Zhang. Revisiting TCGA data identifies key genes in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3292.

Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast.

Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could affect chromosome segregation, DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into non-centromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone with subunits shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. yCAF-1 interacts with Cse4 and can assemble Cse4 nucleosomes in vitro. Loss of yCAF-1 dramatically reduces the amount of Cse4 deposited into chromatin genome-wide when Cse4 is overexpressed. The incorporation of Cse4 genome-wide may have multifactorial effects on growth and gene expression. Loss of yCAF-1 can rescue growth defects and some changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin.

Impact of Mutagens on DNA Replication in Barley Chromosomes.

Replication errors that are caused by mutagens are critical for living cells. The aim of the study was to analyze the distribution of a DNA replication pattern on chromosomes of the H. vulgare ‘Start’ variety using pulse 5-ethynyl-2′-deoxyuridine (EdU) labeling, as well as its relationship to the DNA damage that is induced by mutagenic treatment with maleic hydrazide (MH) and γ ray. To the best of our knowledge, this is the first example of a study of the effects of mutagens on the DNA replication pattern in chromosomes, as well as the first to use EdU labeling for these purposes. The duration of the cell cycle of the Hordeum vulgare ‘Start’ variety was estimated for the first time, as well as the influence of MH and γ ray on it. The distribution of the signals of DNA replication along the chromosomes revealed relationships between DNA replication, the chromatin structure, and DNA damage. MH has a stronger impact on replication than γ ray. Application of EdU seems to be promising for precise analyses of cell cycle disturbances in the future, especially in plant species with small genomes.

Structures of the Catalytic Domain of Bacterial Primase DnaG in Complexes with DNA Provide Insight into Key Priming Events.

Bacterial primase DnaG is an essential nucleic acid polymerase that generates primers for replication of chromosomal DNA. The mechanism of DnaG remains unclear due to the paucity of structural information on DnaG in complexes with other replisome components. Here we report the first crystal structures of noncovalent DnaG-DNA complexes, obtained with the RNA polymerase domain of Mycobacterium tuberculosis DnaG and various DNA ligands. One structure, obtained with ds DNA, reveals interactions with DnaG as it slides on ds DNA and suggests how DnaG binds template for primer synthesis. In another structure, DNA in the active site of DnaG mimics the primer, providing insight into mechanisms for the nucleotide transfer and DNA translocation. In conjunction with the recent cryo-EM structure of the bacteriophage T7 replisome, this study yields a model for primer elongation and hand-off to DNA polymerase.

Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast.

Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could affect chromosome segregation, DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into non-centromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone with subunits shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. yCAF-1 interacts with Cse4 and can assemble Cse4 nucleosomes in vitro. Loss of yCAF-1 dramatically reduces the amount of Cse4 deposited into chromatin genome-wide when Cse4 is overexpressed. The incorporation of Cse4 genome-wide may have multifactorial effects on growth and gene expression. Loss of yCAF-1 can rescue growth defects and some changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin.

DNA replicationmachinery is required for development inDrosophila.

In Drosophila, some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

The function of replication and SCF complex during Drosophila wing development.

Chromosomal DNA replication machinery functions in the growing cells and organs in multicellular organisms. We previously demonstrated that its knockdown in several tissues of Drosophila led to a rough eye phenotype, the loss of bristles in the eye and female sterile. In this paper, I investigated in detail the wing phenotype using RNAi flies, and observed that the knockdown not only of Mcm10 but also of some other prereplicative complex components including Cdt1, Polα-primase, RPA, Psf2 (partner of SLD five 2; a subunit of GINS (Go, Ichi, Nii, and San; five, one, two, and three in Japanese) and Rfc3 (replication factor C 3; a subunit of RFC complex) demonstrated wing phenotypes, using Gal4-driver flies. Surprisingly, some SCF complex components, which control cell cycle progression via protein degradation, also showed the wing phenotype. These results showed that the DNA replication machinery contributes to wing development independently of growth, probably through defects in DNA replication and protein degradation at specific places and times.

DNA replication machinery contributes to development of eye in Drosophila.

Chromosomal DNA replication machinery is essential for growth of all cells in all organs in multicellular organisms. Its knockdown in several tissues of Drosophila led to rough eye phenotype and the loss of bristles in the eye. These results show that the DNA replication machinery contributes to the body development independent of growth.

Do Archaea Need an Origin of Replication?

Chromosomal DNA replication starts at a specific region called an origin of replication. Until recently, all organisms were thought to require origins to replicate their chromosomes. It was recently discovered that some archaeal species do not utilize origins of replication under laboratory growth conditions.