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Specialized DNA polymerases are required in both prokaryotic and eukaryotic cells for bypassing sites of template DNA damage that arrest high-fidelity DNA replication. Recent studies in the literature provide hints of the complexity of DNA switching between polymerases for translesion DNA synthesis (TLS) and those for normal DNA replication.
The Y-family of DNA polymerases are capable of translesion synthesis both in vitro and in vivo. The mouse/human gene (Polkappa/POLK) that encodes the Y-family member Polk is highly expressed in mouse/human testis. Using RT-PCR to amplify the coding sequence of the PolK gene, 11 forms of mouse PolK and 5 forms of human POLK transcripts were identified from testis. This phenomenon is apparently specific to PolK since several other specialized DNA polymerases capable of TLS do not express functional alternative transcripts in the testis. The multiple mouse/human (PolK/POLK) transcripts may encode multiple Polkappa isoforms in testis.
Escherichia coli DNA polymerase IV and V (pol IV and pol V) are error-prone DNA polymerases that are induced as part of the SOS regulon in response to DNA damage. Both are members of the Y-family of DNA polymerases. Their principal biological roles appear to involve translesion synthesis (TLS) and the generation of mutational diversity to cope with stress. Although neither enzyme is known to be involved in base excision repair (BER), we have nevertheless observed apurinic/apyrimidinic 5'-deoxyribose phosphate (AP/5'-dRP) lyase activities intrinsic to each polymerase. Pols IV and V catalyze cleavage of the phosphodiester backbone at the 3'-side of an apurinic/apyrimidinic (AP) site as well as the removal of a 5'-deoxyribose phosphate (dRP) at a preincised AP site. The specific activities of the two error-prone polymerase-associated lyases are approximately 80-fold less than the associated lyase activity of human DNA polymerase beta, which is a key enzyme used in short patch BER. Pol IV forms a covalent Schiff's base intermediate with substrate DNA that is trapped by sodium borohydride, as proscribed by a beta-elimination mechanism. In contrast, a NaBH(4) trapped intermediate is not observed for pol V, even though the lyase specific activity of pol V is slightly higher than that of pol IV. Incubation of pol V (UmuD'(2)C) with a molar excess of UmuD drives an exchange of subunits to form UmuD'D+insoluble UmuC causing inactivation of polymerase and lyase activities. The concomitant loss of both activities is strong evidence that pol V contains a bona fide lyase activity.
One of the least well understood DNA repair processes in cells is the repair of DNA interstrand cross-links (ICLs) which present a major obstacle to DNA replication and must be repaired or bypassed to allow fork progression. Fanconi anemia (FA) is an inherited genome instability syndrome characterized by hypersensitivity to ICL damage. Central to the FA repair pathway is FANCD2 that is mono-ubiquitylated in response to replication stress and ICL damage through the action of the FA core complex and its E3-ubiquitin ligase subunit, FANCL. In its mono-ubiquitylated form FANCD2 is recruited to repair foci where it is believed to somehow coordinate ICL repair and restart of impeded replication forks. However, the precise mechanism through which the FA pathway and mono-ubiquitylation of FANCD2 promotes ICL repair remains unclear. Here we report on a functional homologue of FANCD2 in C. elegans (FCD-2). Although fcd-2 mutants are homozygous viable, they are exquisitely sensitive to ICL-inducing agents, but insensitive to ionizing radiation (IR). fcd-2 is dispensable for meiotic recombination and activation of the S-phase checkpoint, indicating that ICL sensitivity is likely due to a repair rather than a signalling defect. Indeed, we show that FCD-2 is mono-ubiquitylated in response to ICL damage and is recruited to nuclear repair foci. Consistent with the sensitivity of fcd-2 mutants, FCD-2 focus formation is induced in response to ICL damage and replication stress, but not following IR, suggesting that FCD-2 responds to lesions that block DNA replication and not DNA double strand breaks per se. The realization that the FA pathway is conserved in a genetically tractable model system will permit the comprehensive analysis of the interplay between the FA, homologous recombination (HR), translesion synthesis (TLS) and nucleotide excision repair (NER) pathways, critical to the understanding of ICL repair.
In addition to replicative DNA polymerases, cells contain specialized DNA polymerases involved in processes such as lesion tolerance, mutagenesis and immunoglobulin diversity. In Escherichia coli, DNA polymerase V (Pol V), encoded by the umuDC locus, is involved in translesion synthesis (TLS) and mutagenesis. Genetic studies have established that mutagenesis requires both UmuC and a proteolytic product of UmuD (UmuD'). In addition, RecA protein and the replication processivity factor, the beta-clamp, were genetically found to be essential co-factors for mutagenesis. Here, we have reconstituted Pol V-mediated bypass of three common replication-blocking lesions, namely the two major UV-induced lesions and a guanine adduct formed by a chemical carcinogen (G-AAF) under conditions that fulfil these in vivo requirements. Two co-factors are essential for efficient Pol V-mediated lesion bypass: (i) a DNA substrate onto which the beta-clamp is stably loaded; and (ii) an extended single-stranded RecA/ATP filament assembled downstream from the lesion site. For efficient bypass, Pol V needs to interact simultaneously with the beta-clamp and the 3' tip of the RecA filament. Formation of an extended RecA/ATP filament and stable loading of the beta-clamp are best achieved on long single-stranded circular DNA templates. In contrast to previously published data, the single-stranded DNA-binding protein (SSB) is not absolutely required for Pol V-mediated lesion bypass provided ATP, instead of ATPgammaS, activates the RecA filament. Further discrepancies with the existing literature are explainable by the use of either inadequate DNA substrates or a UmuC fusion protein instead of native Pol V.
Y-family DNA polymerases (DNAPs) are a superfamily of evolutionarily related proteins that exist in cells to bypass DNA damage caused by both radiation and chemicals. Cells have multiple Y-family DNAPs, presumably to conduct translesion synthesis (TLS) on DNA lesions of varying structure and conformation. The potent, ubiquitous environmental mutagen/carcinogen benzo[a]pyrene (B[a]P) induces all classes of mutations with G-->T base substitutions predominating. We recently showed that a G-->T mutagenesis pathway for the major adduct of B[a]P ([+ta]-B[a]P-N2-dG) in Escherichia coli depends on Y-family member DNAP V. Since no X-ray crystal study for DNAP V has been reported, no structure is available to help in understanding the structural basis for dATP insertion associated with G-->T mutations from [+ta]-B[a]P-N2-dG. Herein, we do homology modeling to construct a model for UmuC, which is the polymerase subunit of DNAP V. The sequences of eight Y-family DNAPs were aligned based on the positioning of conserved amino acids and an analysis of conserved predicted secondary structure, as well as insights gained from published X-ray structures of five Y-family members. Starting coordinates for UmuC were generated from the backbone coordinates for the Y-family polymerase Dpo4 for reasons discussed, and were refined using molecular dynamics with CHARMM 27. A survey of the literature revealed that E. coli DNAP V and human DNAP eta show a similar pattern of dNTP insertion opposite a variety of DNA lesions. Furthermore, E. coli DNAP IV and human DNAP kappa show a similar dNTP insertional pattern with these same DNA lesions, although the insertional pattern for DNAP IV/kappa differs from the pattern for DNAPs V/eta. These comparisons prompted us to construct and refine models for E. coli DNAP IV and human DNAPs eta and kappa as well. The dNTP/template binding pocket of all four DNAPs was inspected, focusing on the array of seven amino acids that contact the base of the incoming dNTP, as well as the template base. DNAPs V and eta show similarities in this array, and DNAPs IV and kappa also show similarities, although the arrays are different for the two pairs of DNAPs. Thus, there is a correlation between structural similarities and insertional similarities for the pairs DNAPs V/eta and DNAPs IV/kappa. Although the significance of this correlation remains to be elucidated, these observations point the way for future experimental studies.
The potent, ubiquitous environmental mutagen/carcinogen benzo[a]pyrene (B[a]P) induces a single major adduct [+ta]-B[a]P-N2-dG, whose bypass in most cases results in either no mutation (dCTP insertion) or a G-->T mutation (dATP insertion). Translesion synthesis (TLS) of [+ta]-B[a]P-N2-dG generally requires DNA polymerases (DNAPs) in the Y-family, which exist in cells to bypass DNA damage caused by chemicals and radiation. A molecular dynamics (MD) study is described with dCTP opposite [+ta]-B[a]P-N2-dG in Dpo4, which is the best studied Y-family DNAP from a structural point of view. Two orientations of B[a]P-N2-dG (BPmi5 and BPmi3) are considered, along with two orientations of the dCTP (AS1 and AS2), as outlined next. Based on NMR studies, the pyrene moiety of B[a]P-N2-dG is in the minor groove, when paired with dC, and can point toward either the base on the 5'-side (BPmi5) or the 3'-side (BPmi3). Based on published X-ray structures, Dpo4 appears to have two partially overlapping active sites. The architecture of active site 1 (AS1) is similar to all other families of DNAPs (e.g., the shape of the dNTP). Active site 2 (AS2), however, is non-canonical (e.g., the beta- and gamma-phosphates in AS2 are approximately where the alpha- and beta-phosphates are in AS1). In the Dpo4 models generated herein, using the BPmi3 orientation the pyrene moiety of [+ta]-B[a]P-N2-dG points toward the duplex region of the DNA, and is accommodated without distortions in AS1, but with distortions in AS2. Considering the BPmi5 orientation, the pyrene moiety points toward the ss-region of DNA in Dpo4, and sits in a hole defined by the fingers and little fingers domain ("chimney"); BPmi5 is accommodated in AS2 without significant distortions, but poorly in AS1. In summary, when dCTP is paired with [+ta]-B[a]P-N2-dG in the two overlapping active sites in Dpo4, the pyrene in the BPmi3 orientation is accommodated better in active site 1 (AS1), while the pyrene in the BPmi5 orientation is accommodated better in AS2. Finally, we discuss why Y-family DNAPs might have two catalytic active sites.
Accurate quantitation of Magnetic Resonance Spectroscopy (MRS) signals is an essential step before converting the estimated signal parameters, such as frequencies, damping factors, and amplitudes, into biochemical quantities (concentration, pH). Several subspace-based parameter estimators have been developed for this task, which are efficient and accurate time-domain algorithms. However, they suffer from a serious drawback: they allow only a limited inclusion of prior knowledge which is important for accuracy and resolution. In this paper, a new method is presented: KNOB-SVD and its improved variant KNOB-TLS. KNOB-SVD is a recently proposed method, based on the Singular Value Decomposition (SVD), which allows the use of more prior knowledge about the signal parameters than previously published subspace-based methods. We compare its performance in terms of robustness and accuracy with the performance of three commonly used methods for signal parameter estimation: HTLS, a subspace-based method which does not allow any inclusion of prior knowledge, except for the model order; HTLSPK(Delta fd(eq)), a subspace-based method obtained by incorporating in HTLS the prior information that the frequency differences between doublet components are known and the damping factors are equal; and AMARES, an interactive maximum likelihood method that allows the inclusion of a variety of prior knowledge. Extensive simulation and in vivo studies, using (31)P as well as proton MRS signals, show that the new method outperforms HTLS and HTLSPK(Delta fd(eq)) in robustness, accuracy, and resolution, and that it provides parameter estimates comparable to the AMARES ones.
Two new studies provide in vivo ( [this issue of Molecular Cell]) and in vitro ( [this issue of Molecular Cell]) evidence that poleta functions to extend 3' strands exchanged during homologous recombination and raise the issue of how TLS polymerases are selected onto different substrates.
Base excision repair (BER) plays an essential role in protecting cells from mutagenic base damage caused by oxidative stress, hydrolysis, and environmental factors. POLQ is a DNA polymerase, which appears to be involved in translesion DNA synthesis (TLS) past base damage. We disrupted POLQ, and its homologs HEL308 and POLN in chicken DT40 cells, and also created polq/hel308 and polq/poln double mutants. We found that POLQ-deficient mutants exhibit hypersensitivity to oxidative base damage induced by H(2)O(2), but not to UV or cisplatin. Surprisingly, this phenotype was synergistically increased by concomitant deletion of the major BER polymerase, POLbeta. Moreover, extracts from a polq null mutant cell line show reduced BER activity, and POLQ, like POLbeta, accumulated rapidly at sites of base damage. Accordingly, POLQ and POLbeta share an overlapping function in the repair of oxidative base damage. Taken together, these results suggest a role for vertebrate POLQ in BER.
Genomes are damaged by spontaneous decay, chemicals, radiation and replication errors. DNA damage may cause mutations resulting in inheritable disease, cancer and ageing. Oxidative stress from ionising radiation and oxidative metabolism causes base damage, as well as strand breaks in DNA. Base damage is mostly indirect and caused by reactive oxygen species (ROS) generated, e.g. O2(.-) (superoxide radical), OH. (hydroxyl radical) and H2O2 (hydrogen peroxide). ROS also oxidise RNA, lipids, proteins and nucleotides. The first line of defence against ROS is enzymatic inactivation of superoxide by superoxide dismutase and inactivation of the less toxic hydrogen peroxide by catalase. As a second line of defence, incorporation of damaged bases into DNA is prevented by enzymes that hydrolyse oxidised dNTPs (e.g. 8-oxodGTP) to the corresponding dNMP. The third line of defence is repair of oxidative damage in DNA by an intricate network of DNA repair mechanisms. Base excision repair (BER), transcription-coupled repair (TCR), global genome repair (GGR), mismatch repair (MMR), translesion synthesis (TLS), homologous recombination (HR) and non-homologous end-joining (NHEJ) all contribute to repair of oxidative DNA damage. These mechanisms are also integrated with other cellular processes such as cell cycle regulation, transcription and replication and even use some common proteins. BER is the major pathway for repair of oxidative base damage, with TCR and MMR being important backup pathways for repair of transcribed strands and newly replicated strands, respectively. In recent years, several new DNA glycosylases that initiate BER of oxidative damage have been identified. These have specificities overlapping with previously known DNA glycosylases and serve as backups, and may have distinct roles as well. Thus, there is both inter- and intra-pathway complementation in repair of oxidative base damage, explaining the limited effects of absence of single DNA glycosylases in animal model systems.
Hypermutable tandem repeat sequences (TRSs) are present in the genomes of both prokaryotic and eukaryotic organisms. Numerous studies have been conducted in several laboratories over the past decade to investigate the mechanisms responsible for expansions and contractions of microsatellites (a subset of TRSs with a repeat length of 1-6 nucleotides) in the model prokaryotic organism Escherichia coli. Both the frequency of tandem repeat instability (TRI), and the types of mutational events that arise, are markedly influenced by the DNA sequence of the repeat, the number of unit repeats, and the types of cellular pathways that process the TRS. DNA strand slippage is a general mechanism invoked to explain instability in TRSs. Misaligned DNA sequences are stabilized both by favorable base pairing of complementary sequences and by the propensity of TRSs to form relatively stable secondary structures. Several cellular processes, including replication, recombination and a variety of DNA repair pathways, have been shown to interact with such structures and influence TRI in bacteria. This paper provides an overview of our current understanding of mechanisms responsible for TRI in bacteria, with an emphasis on studies that have been carried out in E. coli. In addition, new experimental data are presented, suggesting that TLS polymerases (PolII, PolIV and PolV) do not contribute significantly to TRI in E. coli.
Immune checkpoint blockade has transformed cancer therapy, yet primary and acquired resistance remain major clinical challenges. Increasing evidence indicates that immunotherapy resistance cannot be fully explained by tumor-intrinsic alterations or conventional biomarkers such as PD-L1 expression, tumor mutational burden, or microsatellite instability. Instead, therapeutic response is shaped by the tumor immune microenvironment (TIME) as a heterogeneous, spatially organized, and dynamically evolving ecosystem. Single-cell omics has revealed diverse immune and stromal cell states, including progenitor and terminally exhausted T cells, suppressive myeloid programs, B-cell/TLS-associated immune-reactive states, and CAF-mediated exclusion phenotypes. Spatial transcriptomics, spatial proteomics, and imaging-based approaches further demonstrate that these cell states assemble into distinct immune niches, including immune-inflamed, T-cell-excluded, myeloid-suppressive, metabolic/hypoxic, and TLS-associated niches. These spatial ecosystems determine whether antitumor immune cells can access malignant cells, receive antigen-presenting support, or become restrained by stromal, vascular, metabolic, and myeloid barriers. In this review, we summarize how single-cell and spatial multi-omics redefine TIME heterogeneity in immunotherapy resistance, highlight ligand-receptor communication networks linking cell states to spatial immune dysfunction, and discuss emerging translational biomarkers for patient stratification. We further propose that future immunotherapy biomarkers should evolve from static single-marker assays toward longitudinal, spatially resolved, and interpretable multi-omics models that guide precision combination immunotherapy.
RNA transport is an important and fundamental event for local protein synthesis, especially in neurons. RNA is transported as large granules, but little is known about them. Here, we isolated a large RNase-sensitive granule (size: 1000S approximately) as a binding partner of conventional kinesin (KIF5). We identified a total of 42 proteins with mRNAs for CaMKIIalpha and Arc in the granule. Seventeen of the proteins (hnRNP-U, Pur alpha and beta, PSF, DDX1, DDX3, SYNCRIP, TLS, NonO, HSPC117, ALY, CGI-99, staufen, three FMRPs, and EF-1alpha) were extensively investigated, including their classification, binding combinations, and necessity for the "transport" of RNA. These proteins and the mRNAs were colocalized to the kinesin-associated granules in dendrites. The granules moved bidirectionally, and the distally directed movement was enhanced by the overexpression of KIF5 and reduced by its functional blockage. Thus, kinesin transports RNA via this granule in dendrites coordinately with opposite motors, such as dynein.
The genomic RNA of Turnip yellow mosaic virus (TYMV) has an 82-nucleotide-long tRNA-like structure at its 3'-end that can be valylated and then form a stable complex with translation elongation factor eEF1A.GTP. Transcription of this RNA by TYMV RNA-dependent RNA polymerase (RdRp) to yield minus strands has previously been shown to initiate within the 3'-CCA sequence. We have now demonstrated that minus strand synthesis is strongly repressed upon the binding of eEF1A.GTP to the valylated viral RNA. eEF1A.GTP had no effect on RNA synthesis templated by non-aminoacylated RNA. Higher eEF1A.GTP levels were needed to repress minus strand synthesis templated by valyl-EMV TLS RNA, which binds eEF1A.GTP with lower affinity than does valyl-TYMV RNA. Repression by eEF1A.GTP was also observed with a methionylated variant of TYMV RNA and with aminoacylated tRNAHis, tRNAAla, and tRNAPhe transcripts. It is proposed that minus strand repression by eEF1A.GTP binding occurs early during infection to help coordinate the competing translation and replication functions of the genomic RNA.
The composition of natural organic matter (NOM) fractions before and after the reaction with chlorine dioxide as well as ozone was studied by means of total luminescence spectroscopy (TLS) and synchronous scanning fluorescence measurements. The excitation-emission matrices spectra (EEMs) of natural as well as oxidised NOM fractions revealed two well-resolved bands with maxima at Ex/Em of 250-265/422-452 nm and at Ex/Em 300-336/414-446 nm ascribed to humic and fulvic material. The study of emission and synchronous spectra also confirmed the presence of protein-like constituents in all examined NOM fractions. The study of EEMs proved, that oxidation of all NOM fractions with ClO(2) caused mainly the break-up of molecules into smaller fragments and a decrease of the aromaticity. Changes in EEMs after the oxidation of individual NOM fractions with O(3) confirmed the formation of a significant amount of ozonation by-products, i.e. carboxylic acids, aldehydes and ketones during the oxidation process. In addition, the fluorescence studies confirmed relatively high reactivity of all NOM fractions with ClO(2) as well as with ozone.
In the anaerobic respiration of sulfate, performed by sulfate-reducing prokaryotes, reduction of the terminal electron acceptor takes place in the cytoplasm. The membrane-associated electron transport chain that feeds electrons to the cytoplasmic reductases is still very poorly characterized. In this study we report the isolation and characterization of a novel membrane-bound redox complex from Desulfovibrio desulfuricans ATCC 27774. This complex is formed by three subunits, and contains two hemes b, two FAD groups and several iron-sulfur centers. The two hemes b are low-spin, with macroscopic redox potentials of +75 and -20 mV at pH 7.6. Both hemes are reduced by menadiol, a menaquinone analogue, indicating a function for this complex in the respiratory electron-transport chain. EPR studies of the as-isolated and dithionite-reduced complex support the presence of a [3Fe-4S](1+/0) center and at least four [4Fe-4S](2+/1+) centers. Cloning of the genes coding for the complex subunits revealed that they form a putative transcription unit and have homology to subunits of heterodisulfide reductases (Hdr). The first and second genes code for soluble proteins that have homology to HdrA, whereas the third gene codes for a novel type of membrane-associated protein that contains both a hydrophobic domain with homology to the heme b protein HdrE and a hydrophilic domain with homology to the iron-sulfur protein HdrC. Homologous operons are found in the genomes of other sulfate-reducing organisms and in the genome of the green-sulfur bacterium Chlorobium tepidum TLS. The isolated complex is the first example of a new family of respiratory complexes present in anaerobic prokaryotes.
The dynamic behavior of proteins in crystals is examined by comparing theory and experiments. The Gaussian network model (GNM) and a simplified version of the crystallographic translation libration screw (TLS) model are used to calculate mean square fluctuations of C(alpha) atoms for a set of 113 proteins whose structures have been determined by x-ray crystallography. Correlation coefficients between the theoretical estimations and experiment are calculated and compared. The GNM method gives better correlation with experimental data than the rigid-body libration model and has the added benefit of being able to calculate correlations between the fluctuations of pairs of atoms. By incorporating the effect of neighboring molecules in the crystal the correlation is further improved.