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Gene Oct 2023Regulation of the cell cycle process is an effective measure to ensure the stability and fidelity of genetic material during the reproduction of bacteria under different...
Regulation of the cell cycle process is an effective measure to ensure the stability and fidelity of genetic material during the reproduction of bacteria under different stresses. The small RNA DsrA helps bacteria adapt to environments by binding to multiple targets, but its association with the cell cycle remains unclear. Detection by flow cytometry, we first found that the knockout of dsrA promoted replication initiation, and corresponding overexpression of DsrA inhibited replication initiation in Escherichia coli. The absence of the chaperone protein Hfq, the DNA replication negative regulator protein Dps, or the transcription factor OxyR, was found to cause DsrA to no longer inhibit replication initiation. Excess DsrA promotes expression of the oxyR and dps gene, whereas β-galactosidase activity assay showed that deleting oxyR limited the enhancement of dps promoter transcriptional activity by DsrA. OxyR is a known positive regulator of Dps. Our data suggests that the effect of DsrA on replication initiation requires Hfq and that the upregulation of Dps expression by OxyR in response to DsrA levels may be a potential regulatory pathway for the negative regulation of DNA replication initiation.
Topics: Escherichia coli; RNA; Escherichia coli Proteins; Bacterial Proteins; DNA Replication; Gene Expression Regulation, Bacterial; RNA, Small Untranslated
PubMed: 37482259
DOI: 10.1016/j.gene.2023.147659 -
NAR Cancer Sep 2023It has been over a decade since the initial identification of exonuclease domain mutations in the genes encoding the catalytic subunits of replication DNA polymerases ϵ...
It has been over a decade since the initial identification of exonuclease domain mutations in the genes encoding the catalytic subunits of replication DNA polymerases ϵ and δ ( and ) in tumors from highly mutated endometrial and colorectal cancers. Interest in studying and has increased significantly since then. Prior to those landmark cancer genome sequencing studies, it was well documented that mutations in replication DNA polymerases that reduced their DNA synthesis accuracy, their exonuclease activity or their interactions with other factors could lead to increased mutagenesis, DNA damage and even tumorigenesis in mice. There are several recent, well-written reviews of replication DNA polymerases. The aim of this review is to gather and review in some detail recent studies of DNA polymerases ϵ and δ as they pertain to genome instability, cancer and potential therapeutic treatments. The focus here is primarily on recent informative studies on the significance of mutations in genes encoding their catalytic subunits ( and ), mutational signatures, mutations in associated genes, model organisms, and the utility of chemotherapy and immune checkpoint inhibition in polymerase mutant tumors.
PubMed: 37388540
DOI: 10.1093/narcan/zcad033 -
Journal of Molecular Biology Dec 2023The Y-family DNA polymerases - Pol ι, Pol η, Pol κ and Rev1 - are most well-known for their roles in the DNA damage tolerance pathway of translesion synthesis (TLS)....
The Y-family DNA polymerases - Pol ι, Pol η, Pol κ and Rev1 - are most well-known for their roles in the DNA damage tolerance pathway of translesion synthesis (TLS). They function to overcome replication barriers by bypassing DNA damage lesions that cannot be normally replicated, allowing replication forks to continue without stalling. In this work, we demonstrate a novel interaction between each Y-family polymerase and the nucleotide excision repair (NER) proteins, RAD23A and RAD23B. We initially focus on the interaction between RAD23A and Pol ι, and through a series of biochemical, cell-based, and structural assays, find that the RAD23A ubiquitin-binding domains (UBA1 and UBA2) interact with separate sites within the Pol ι catalytic domain. While this interaction involves the ubiquitin-binding cleft of UBA2, Pol ι interacts with a distinct surface on UBA1. We further find that mutating or deleting either UBA domain disrupts the RAD23A-Pol ι interaction, demonstrating that both interactions are necessary for stable binding. We also provide evidence that both RAD23 proteins interact with Pol ι in a similar manner, as well as with each of the Y-family polymerases. These results shed light on the interplay between the different functions of the RAD23 proteins and reveal novel binding partners for the Y-family TLS polymerases.
Topics: DNA Damage; DNA Polymerase iota; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Ubiquitins; DNA-Binding Proteins; DNA Repair Enzymes
PubMed: 37935254
DOI: 10.1016/j.jmb.2023.168353 -
Proceedings of the National Academy of... Dec 2023DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA...
DNA replication in all cells begins with the melting of base pairs at the duplex origin to allow access to single-stranded DNA templates which are replicated by DNA polymerases. In bacteria, origin DNA is presumed to be melted by accessory proteins that allow loading of two ring-shaped replicative helicases around single-strand DNA (ssDNA) for bidirectional unwinding and DNA replication. In eukaryotes, by contrast, two replicative CMG (Cdc45-Mcm2-7-GINS) helicases are initially loaded head to head around origin double-strand DNA (dsDNA), and there does not appear to be a separate origin unwinding factor. This led us to investigate whether head-to-head CMGs use their adenosine triphosphate (ATP)-driven motors to initiate duplex DNA unwinding at the origin. Here, we show that CMG tracks on one strand of the duplex while surrounding it, and this feature allows two head-to-head CMGs to unwind dsDNA by using their respective motors to pull on opposite strands of the duplex. We further show that while CMG is capable of limited duplex unwinding on its own, the extent of unwinding is greatly and rapidly stimulated by addition of the multifunctional CMG-binding protein Mcm10 that is critical for productive initiation of DNA replication in vivo. On the basis of these findings, we propose that Mcm10 is a processivity or positioning factor that helps translate the work performed by the dual CMG motors at the origin into productive unwinding that facilitates bidirectional DNA replication.
Topics: Minichromosome Maintenance Proteins; Saccharomyces cerevisiae Proteins; DNA Replication; DNA; DNA, Single-Stranded
PubMed: 38109526
DOI: 10.1073/pnas.2316466120 -
Climacteric : the Journal of the... Oct 2023Premature ovarian insufficiency (POI) is characterized by a loss of regular hormone production and egg release in women below the age of 40 years, which often leads to...
Premature ovarian insufficiency (POI) is characterized by a loss of regular hormone production and egg release in women below the age of 40 years, which often leads to infertility, vaginal dryness and dysfunctional sleep. Acknowledging the common co-occurrence of insomnia and POI, we tested the overlap between POI and insomnia-associated genes, which were implicated in previous large-scale populational genetics efforts. Among the 27 overlapping genes, three pathways were found as enriched: DNA replication, homologous recombination and Fanconi anemia. We then describe biological mechanisms, which link these pathways to a dysfunctional regulation and response to oxidative stress. We propose that oxidative stress may correspond to one of the convergent cellular processes between ovarian malfunction and insomnia pathogenic etiology. This overlap might also be driven by cortisol release associated with dysregulated DNA repair mechanisms. Benefiting from the enormous advances in populational genetics studies, this study provides a novel outlook on the relationship between insomnia and POI. The shared genetic factors and critical biological nodes between these two comorbidities may lead to identification of putative pharmacological and therapeutical targets, which can leverage novel approaches to treat or alleviate their symptoms.
Topics: Female; Humans; Adult; Sleep Initiation and Maintenance Disorders; Primary Ovarian Insufficiency; Ovarian Diseases; Menopause, Premature
PubMed: 37144421
DOI: 10.1080/13697137.2023.2205580 -
BioRxiv : the Preprint Server For... Dec 2023Mammalian genomes are replicated in a precise order during S phase, which is cell-type-specific and correlates with local transcriptional activity, chromatin...
Mammalian genomes are replicated in a precise order during S phase, which is cell-type-specific and correlates with local transcriptional activity, chromatin modifications and chromatin architecture. However, the causal relationships between these features and the key regulators of DNA replication timing (RT) are largely unknown. Here, machine learning was applied to quantify chromatin features, including epigenetic marks, histone variants and chromatin architectural factors, best predicting local RT under steady-state and RT changes during early embryonic stem (ES) cell differentiation. About one-third of genome exhibited RT changes during the differentiation. Combined, chromatin features predicted steady-state RT and RT changes with high accuracy. Of these features, histone H3 lysine 4 monomethylation (H3K4me1) catalyzed by MLL3/4 (also known as KMT2C/D) emerged as a top predictor. Loss of (but not ) or their enzymatic activity resulted in erasure of genome-wide RT dynamics during ES cell differentiation. Sites that normally gain H3K4me1 in a MLL3/4-dependent fashion during the transition failed to transition towards earlier RT, often with transcriptional activation unaffected. Further analysis revealed a requirement for MLL3/4 in promoting DNA replication initiation zones through MCM2 recruitment, providing a direct link for its role in regulating RT. Our results uncover MLL3/4-dependent H3K4me1 as a functional regulator of RT and highlight a causal relationship between the epigenome and RT that is largely uncoupled from transcription. These findings uncover a previously unknown role for MLL3/4-dependent chromatin functions which is likely relevant to the numerous diseases associated with MLL3/4 mutations.
PubMed: 38106216
DOI: 10.1101/2023.12.07.569680 -
BioRxiv : the Preprint Server For... Mar 2024Cohesin is required for chromatin loop formation. However, its precise role in regulating gene transcription remains largely unknown. We investigated the relationship...
Cohesin is required for chromatin loop formation. However, its precise role in regulating gene transcription remains largely unknown. We investigated the relationship between cohesin and RNA Polymerase II (RNAPII) using single-molecule mapping and live-cell imaging methods in human cells. Cohesin-mediated transcriptional loops were highly correlated with those of RNAPII and followed the direction of gene transcription. Depleting RAD21, a subunit of cohesin, resulted in the loss of long-range (>100 kb) loops between distal (super-)enhancers and promoters of cell-type-specific genes. By contrast, the short-range (<50 kb) loops were insensitive to RAD21 depletion and connected genes that are mostly housekeeping. This result explains why only a small fraction of genes are affected by the loss of long-range chromatin interactions due to cohesin depletion. Remarkably, RAD21 depletion appeared to up-regulate genes located in early initiation zones (EIZ) of DNA replication, and the EIZ signals were amplified drastically without RAD21. Our results revealed new mechanistic insights of cohesin's multifaceted roles in establishing transcriptional loops, preserving long-range chromatin interactions for cell-specific genes, and maintaining timely order of DNA replication.
PubMed: 38585764
DOI: 10.1101/2024.03.25.586715 -
Anais Da Academia Brasileira de Ciencias 2023Chicken broilers digestibility and performance fed with different ME levels, with and without adjustments of digestible lysine, calcium, and available phosphorus, were...
Chicken broilers digestibility and performance fed with different ME levels, with and without adjustments of digestible lysine, calcium, and available phosphorus, were evaluated. For digestibility, 210 male Cobb 500 chicken broilers were used and distributed into a 3x2+1 factorial arrangement, with three ME levels (3050; 3125 and 3200 kcal/kg) with and without nutrient adjustment, plus one control treatment (2975 kcal ME/kg), totaling seven treatments including six repetitions with five birds into each repetition. For initial performance, 1120 birds were distributed randomly with eight replications within treatments and 20 birds for each replication. For final performance, 1008 chickens were distributed with eight replications and 18 birds for each replication. The DCDM and DCCP were improved (P<0.05) according to the increase of ME and the adjustment in dietary nutrients, as well as GE digestibility. The final performance showed no interaction (P>0.05) between energy and nutrient adjustment, but the increase in energy levels improved the feed conversion ratio (FCR=1.370). Increasing energy density with nutrient adjustment improves both nutrient utilization and bird performance.
Topics: Animals; Male; Calcium; Chickens; Lysine; Nutrients; Phosphorus
PubMed: 37729298
DOI: 10.1590/0001-3765202320191391 -
Microbiology and Molecular Biology... Dec 2023SUMMARYRecBCD enzyme is a multi-functional protein that initiates the major pathway of homologous genetic recombination and DNA double-strand break repair in . It is... (Review)
Review
SUMMARYRecBCD enzyme is a multi-functional protein that initiates the major pathway of homologous genetic recombination and DNA double-strand break repair in . It is also required for high cell viability and aids proper DNA replication. This 330-kDa, three-subunit enzyme is one of the fastest, most processive helicases known and contains a potent nuclease controlled by Chi sites, hotspots of recombination, in DNA. RecBCD undergoes major changes in activity and conformation when, during DNA unwinding, it encounters Chi (5'-GCTGGTGG-3') and nicks DNA nearby. Here, we discuss the multitude of mutations in each subunit that affect one or another activity of RecBCD and its control by Chi. These mutants have given deep insights into how the multiple activities of this complex enzyme are coordinated and how it acts in living cells. Similar studies could help reveal how other complex enzymes are controlled by inter-subunit interactions and conformational changes.
Topics: Exodeoxyribonuclease V; Recombination, Genetic; DNA Helicases; DNA Repair; Escherichia coli; DNA; Escherichia coli Proteins
PubMed: 38047637
DOI: 10.1128/mmbr.00041-23 -
Nucleic Acids Research Oct 2023For DNA replication initiation in Bacteria, replication initiation proteins bind to double-stranded DNA (dsDNA) and interact with single-stranded DNA (ssDNA) at the...
For DNA replication initiation in Bacteria, replication initiation proteins bind to double-stranded DNA (dsDNA) and interact with single-stranded DNA (ssDNA) at the replication origin. The structural-functional relationship of the nucleoprotein complex involving initiator proteins is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), the analysis of mutant proteins and crystal structures, we defined amino acid residues essential for the interaction between plasmid Rep proteins, TrfA and RepE, and ssDNA. This interaction and Rep binding to dsDNA could not be provided in trans, and both are important for dsDNA melting at DNA unwinding element (DUE). We solved two crystal structures of RepE: one in a complex with ssDNA DUE, and another with both ssDNA DUE and dsDNA containing RepE-specific binding sites (iterons). The amino acid residues involved in interaction with ssDNA are located in the WH1 domain in stand β1, helices α1 and α2 and in the WH2 domain in loops preceding strands β1' and β2' and in these strands. It is on the opposite side compared to RepE dsDNA-recognition interface. Our data provide evidence for a loop-back mechanism through which the plasmid replication initiator molecule accommodates together dsDNA and ssDNA.
Topics: DNA, Single-Stranded; DNA-Binding Proteins; DNA Replication; Plasmids; DNA; Amino Acids
PubMed: 37713613
DOI: 10.1093/nar/gkad740