GC-rich Sequences Research Articles

Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD

Expanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G4C2 repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G4C2 repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G4C2 repeats in human C9ORF72-ALS/FTD neurons.

The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog.

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

The Nuclear Receptor COUP-TFII Regulates Amhr2 Gene Transcription via a GC-Rich Promoter Element in Mouse Leydig Cells.

The nuclear receptor chicken ovalbumin upstream promoter–transcription factor type II (COUP-TFII)/NR2F2 is expressed in adult Leydig cells, and conditional deletion of the Coup-tfii/Nr2f2 gene impedes their differentiation. Steroid production is also reduced in COUP-TFII–depleted Leydig cells, supporting an additional role in steroidogenesis for this transcription factor. COUP-TFII action in Leydig cells remains to be fully characterized. In the present work, we report that COUP-TFII is an essential regulator of the gene encoding the anti-Müllerian hormone receptor type 2 (Amhr2), which participates in Leydig cell differentiation and steroidogenesis. We found that Amhr2 mRNA levels are reduced in COUP-TFII–depleted MA-10 Leydig cells. Consistent with this, COUP-TFII directly activates a −1486 bp fragment of the mouse Amhr2 promoter in transient transfection assays. The COUP-TFII responsive region was localized between −67 and −34 bp. Chromatin immunoprecipitation assay confirmed COUP-TFII recruitment to the proximal Amhr2 promoter whereas DNA precipitation assay revealed that COUP-TFII associates with the −67/−34 bp region in vitro. Even though the −67/−34 bp region contains an imperfect nuclear receptor element, COUP-TFII–mediated activation of the Amhr2 promoter requires a GC-rich sequence at −39 bp known to bind the specificity protein (SP)1 transcription factor. COUP-TFII transcriptionally cooperates with SP1 on the Amhr2 promoter. Mutations that altered the GCGGGGCGG sequence at −39 bp abolished COUP-TFII–mediated activation, COUP-TFII/SP1 cooperation, and reduced COUP-TFII binding to the proximal Amhr2 promoter. Our data provide a better understanding of the mechanism of COUP-TFII action in Leydig cells through the identification and regulation of the Amhr2 promoter as a novel target.

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a

The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double-strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell-specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20-80-fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non-homologous end-joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns.

Novel probes for label-free detection of neurodegenerative GGGGCC repeats associated with amyotrophic lateral sclerosis.

DNA repeat expansion sequences cause a myriad of neurological diseases when they expand beyond a critical threshold. Previous electrochemical approaches focused on the detection of trinucleotide repeats (CAG, CGG, and GAA) and relied on labeling of the probe and/or target strands or enzyme-linked assays. However, detection of expanded GC-rich sequences is challenging because they are prone to forming secondary structures such as cruciforms and quadruplexes. Here, we present label-free detection of hexanucleotide GGGGCC repeat sequences, which cause the leading genetic form of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The approach relies on capturing targets by surface-bound oligonucleotide probes with a different number of complementary repeats, which proportionately translates the length of the target strands into charge transfer resistance (RCT) signal measured by electrochemical impedance spectroscopy. The probe carrying three tandem repeats transduces the number of repeats into RCT with a 3× higher calibration sensitivity and detection limit. Chronocoulometric measurements show a decrease in surface density with increasing repeat length, which is opposite of the impedance trend. This implies that the length of the target itself can contribute to amplification ofthe impedance signal independent of thesurface density. Moreover, the probe can distinguish between a control and patient sequences while remaining insensitive to non-specific Huntington's disease (CAG) repeats in the presence of a complementary target. This label-free strategy might be applied to detect the length of other neurodegenerative repeat sequences using short probes with a few complementary repeats. Graphical abstract Short oligomeric probes with multiple complementary repeats detect long neurodegenerative targets with high sensitivity and transduce into higher impedance signal.

Vascular alteration in relation to fosfomycine: In silico and in vivo investigations using a chick embryo model

Fosfomycin residues are found in the egg following administration in the layer hen. In this regard, some aspects of embryo-toxicity of fosfomycin have been documented previously. The exact mechanism by which fosfomycin causes embryo-toxicity is not clearly understood. We hypothesis that fosfomycin may alter vasculature as well as normal expression of genes, which are associated with vascular development. Therefore, the present study aimed to address these issues through in silico and in vivo investigations. At first, embryo-toxicity and anti-angiogenic effects of fosfomycin were tested using computerized programs. After that, fertile chicken eggs were treated with fosfomycin and chorioallantoic membrane vasculature was assessed through morphometric, molecular and histopathological assays. The results showed that fosfomycin not only interacted with VEGF-A protein and promoter, but also altered embryonic vasculature and decreased expression level of VEGF-A. Reticulin staining of treated group was also confirmed decreased vasculature. The minor groove of DNA was the preferential binding site for fosfomycin with its selective binding to GC-rich sequences. We suggested that the affinity of fosfomycin for VEGF-A protein and promoter as well as alteration of the angiogenic signaling pathway may cause vascular damage during embryonic growth. Hence, veterinarians should be aware of such effects and limit the use of this drug during the developmental stages of the embryo, particularly in breeder farms. Considering the anti-angiogenic activity and sequence selectivity of fosfomycin, a major advantage that seems to be very promising is the fact that it is possible to achieve a sequence-selective binding drug for cancer.

Neutralized chimeric DNA probe for the improvement of GC-rich RNA detection specificity on the nanowire field-effect transistor

Silicon nanowire (SiNW) field-effect transistors (FETs) is a powerful tool in genetic molecule analysis because of their high sensitivity, short detection time, and label-free detection. In nucleic acid detection, GC-rich nucleic acid sequences form self- and cross-dimers and stem-loop structures, which can easily obtain data containing signals from nonspecific DNA binding. The features of GC-rich nucleic acid sequences cause inaccuracies in nucleic acid detection and hinder the development of precision medicine. To improve the inaccurate detection results, we used phosphate-methylated (neutral) nucleotides to synthesize the neutralized chimeric DNA oligomer probe. The probe fragment originated from a primer for the detection of hepatitis C virus (HCV) genotype 3b, and single-mismatched and perfect-matched targets were designed for single nucleotide polymorphisms (SNP) detection on the SiNW FET device. Experimental results revealed that the HCV-3b chimeric neutralized DNA (nDNA) probe exhibited better performance for SNP discrimination in 10 mM bis-tris propane buffer at 25 °C than a regular DNA probe. The SNP discrimination of the nDNA probe could be further improved at 40 °C on the FET device. Consequently, the neutralized chimeric DNA probe could successfully distinguish SNP in the detection of GC-rich target sequences under optimal operating conditions on the SiNW FET device.

Abstract 9: Effect of C8-side chain on the cytotoxicity and NF-kB inhibitory capacity of pyrrolobenzodiazepines

Abstract Pyrrolobenzodiazepines (PBDs) have long been of interest in chemotherapeutic research as potential clinical agents. Recently, the conjugation of non-covalently interactive moieties to the C8-position of the PBD structure via a linker of variable length has emerged as a strategy to enhance the DNA sequence specificity of PBDs. This has led to the development of highly cytotoxic agents, which also act as inhibitors of transcription factors such as NF-κB. For example, GWL-78, a C8-linked PBD-Py-Py (Py = pyrrole) conjugate, has been shown to block interaction of the transcription factor NF-Y, and KMR-28-39, a C8-linked PBD-MPB conjugate with an affinity for GC-rich sequences of DNA inhibits activity of the transcription factor NF-κB. As up-regulation of NF-κB has been implicated in the development and progression of several cancer types, this may contribute to the high potency observed for PBD monomers such as KMR-28-39 (i.e., femtomolar IC50 values in some cell lines). In this study, the relationship between the cytotoxicity and transcription factor inhibition of a series of C8-linked PBD hybrid structures was explored. The systematic shortening of the non-covalent element of a C8-linked PBD conjugate, DC-1-92 led to the synthesis of a 19-member library of novel C8-PBD monomers. The critical elements of DC-1-92, which were required to render the molecule cytotoxic, were elucidated by an annexin V assay. The effects of shortening the non-covalent element of the molecule on transcription factor inhibitory capacity were also explored through an ELISA-based measurement of nuclear NF-κB subunit DNA binding following exposure of JJN3 cells to the synthesised molecules. While shortening the non-covalently interactive component of DC-1-92 had less of an effect than expected upon cytotoxicity, the shortening notably reduced the NF-κB inhibitory activity of the shortened analogues. DC-1-92, containing the longest non-covalently interactive C8-element, displayed the most effective inhibition of NF-κB, and effects upon nuclear p65, and RelB levels appeared to diminish in tandem with the shortening of the length of the C8 side chain. These data suggest that the length of the C8 side chain of PBD hybrid structures impacts upon the DNA binding of both canonical and non-canonical NF-κB subunits but this is independent of their cytotoxic effects. Citation Format: David B. Corcoran, Thomas Lewis, Kazi S. Nahar, Christopher Fegan, Chris Pepper, David E. Thurston, Khondaker Miraz Rahman. Effect of C8-side chain on the cytotoxicity and NF-kB inhibitory capacity of pyrrolobenzodiazepines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 9.

A massively parallel 3' UTR reporter assay reveals relationships between nucleotide content, sequence conservation, and mRNA destabilization.

Compared to coding sequences, untranslated regions of the transcriptome are not well conserved, and functional annotation of these sequences is challenging. Global relationships between nucleotide composition of 3′ UTR sequences and their sequence conservation have been appreciated since mammalian genomes were first sequenced, but the functional relevance of these patterns remain unknown. We systematically measured the effect on gene expression of the sequences of more than 25,000 RNA-binding protein (RBP) binding sites in primary mouse T cells using a massively parallel reporter assay. GC-rich sequences were destabilizing of reporter mRNAs and come from more rapidly evolving regions of the genome. These sequences were more likely to be folded in vivo and contain a number of structural motifs that reduced accumulation of a heterologous reporter protein. Comparison of full-length 3′ UTR sequences across vertebrate phylogeny revealed that strictly conserved 3′ UTRs were GC-poor and enriched in genes associated with organismal development. In contrast, rapidly evolving 3′ UTRs tended to be GC-rich and derived from genes involved in metabolism and immune responses. Cell-essential genes had lower GC content in their 3′ UTRs, suggesting a connection between unstructured mRNA noncoding sequences and optimal protein production. By reducing gene expression, GC-rich RBP-occupied sequences act as a rapidly evolving substrate for gene regulatory interactions.

Egr-1 deficiency protects host against Pseudomonas aeruginosa lung infection

Abstract Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of nosocomial infections in cystic fibrosis patients and immunocompromised individuals. The molecular mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Early Growth Response 1 (Egr-1) is a zinc-finger transcription factor that binds to the GC-rich DNA consensus sequences in the promotor of target genes, important for cell growth, differentiation and survive. Aberrant expression of Egr-1 has been implicated in many inflammatory diseases. In this study, we demonstrate that Egr-1 deficiency protects host against P. aeruginosa infection in a mouse model of acute bacterial pneumonia. Egr-1 expression was rapidly and transiently induced both in vitro and in vivo upon P. aeruginosa infection. Egr-1-deficient mice displayed decreased disease score, reduced systemic levels of proinflammatory cytokines and impaired NF-κB and NFAT activation compared to wild-type mice. Interestingly, Egr-1 deficiency leads to enhanced bacterial clearance and increased nitric oxide production in lung whereas it has no impact on neutrophil recruitment. Further studies revealed that Egr-1-deficient neutrophils displayed elevated bacterial killing ability. Altogether, these findings suggest that Egr-1 promotes inflammatory responses by enhancing NF-κB and NFAT activation and plays a detrimental role in host defense against P. aeruginosa lung infection by negatively regulating nitric oxide production for bacterial clearance.

The genome-wide sequence preference of ionising radiation-induced cleavage in human DNA.

For ionising radiation (IR)-induced cellular toxicity, DNA cleavage is thought to be a crucial step. In this paper, the genome-wide DNA sequence preference of gamma radiation-induced cleavage was investigated in purified human DNA. We utilised Illumina short read technology and over 80 million double-strand breaks (DSBs) were analysed in this study. The frequency of occurrence of individual nucleotides at the 50,000 most frequently cleaved sites was calculated and C nucleotides were found to be most prevalent at the cleavage site, followed by G and T, with A being the least prevalent. 5'-C*C and 5'-CC* dinucleotides (where * is the cleavage site) were found to be the present at the highest frequency at the cleavage site; while it was 5'-CC*C for trinucleotides and 5'-GCC*C and 5'-CC*CC for tetranucleotides. The frequency of occurrence of individual nucleotides at the most frequently cleaved sites was determined and the nucleotides in the sequence 5'-GGC*MH (where M is A or C, H is any nucleotide except G) were found to occur most frequently for DNA that was treated with endonuclease IV (to remove blocking 3'-phosphoglycolate termini); and 5'-GSC*MH (where S is G or C) for non-endonuclease IV-treated DNA. It was concluded that GC-rich sequences were preferentially targeted for cleavage by gamma irradiation. This was the first occasion that an extensive examination of the genome-wide DNA sequence preference of IR-induced DSBs has been performed.

Intragenomic heterogeneity of intergenic ribosomal DNA spacers in Cucurbita moschata is determined by DNA minisatellites with variable potential to form non-canonical DNA conformations.

The intergenic spacer (IGS) of rDNA is frequently built of long blocks of tandem repeats. To estimate the intragenomic variability of such knotty regions, we employed PacBio sequencing of the Cucurbita moschata genome, in which thousands of rDNA copies are distributed across a number of loci. The rRNA coding regions are highly conserved, indicating intensive interlocus homogenization and/or high selection pressure. However, the IGS exhibits high intragenomic structural diversity. Two repeated blocks, R1 (300–1250 bp) and R2 (290–643 bp), account for most of the IGS variation. They exhibit minisatellite-like features built of multiple periodically spaced short GC-rich sequence motifs with the potential to adopt non-canonical DNA conformations, G-quadruplex-folded and left-handed Z-DNA. The mutual arrangement of these motifs can be used to classify IGS variants into five structural families. Subtle polymorphisms exist within each family due to a variable number of repeats, suggesting the coexistence of an enormous number of IGS variants. The substantial length and structural heterogeneity of IGS minisatellites suggests that the tempo of their divergence exceeds the tempo of the homogenization of rDNA arrays. As frequently occurring among plants, we hypothesize that their instability may influence transcription regulation and/or destabilize rDNA units, possibly spreading them across the genome.

Identification and Characterization of oriT and Two Mobilization Genes Required for Conjugative Transfer of Salmonella Genomic Island 1.

The integrative mobilizable elements of SGI1-family considerably contribute to the spread of resistance to critically important antibiotics among enteric bacteria. Even though many aspects of SGI1 mobilization by IncA and IncC plasmids have been explored, the basic transfer elements such as oriT and self-encoded mobilization proteins remain undiscovered. Here we describe the mobilization region of SGI1 that is well conserved throughout the family and carries the oriTSGI1 and two genes, mpsA and mpsB (originally annotated as S020 and S019, respectively) that are essential for the conjugative transfer of SGI1. OriTSGI1, which is located in the vicinity of the two mobilization genes proved to be a 125-bp GC-rich sequence with several important inverted repeat motifs. The mobilization proteins MpsA and MpsB are expressed from a bicistronic mRNA, although MpsB can be produced from its own mRNA as well. The protein structure predictions imply that MpsA belongs to the lambda tyrosine recombinase family, while MpsB resembles the N-terminal core DNA binding domains of these enzymes. The results suggest that MpsA may act as an atypical relaxase, which needs MpsB for SGI1 transfer. Although the helper plasmid-encoded relaxase proved not to be essential for SGI1 transfer, it appeared to be important to achieve the high transfer rate of the island observed with the IncA/IncC-SGI1 system.

Nanomechanics of Diaminopurine-Substituted DNA

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensions. Here, we show that DAP-DNA overstretches at 52 pN. In summary, DAP substitution decreases the overall stability of the B-form double helix, biasing toward non-B-form DNA helix conformations at zero tension and facilitating the B-to-S transition at high tension.

Architectural roles of Cren7 in folding crenarchaeal chromatin filament.

Archaea have evolved various strategies in chromosomal organization. While histone homologues exist in most archaeal phyla, Cren7 is a chromatin protein conserved in the Crenarchaeota. Here, we show that Cren7 preferentially binds DNA with AT-rich sequences over that with GC-rich sequences with a binding size of 6~7bp. Structural studies of Cren7 in complex with either an 18-bp or a 20-bp double-stranded DNA fragment reveal that Cren7 binds to the minor groove of DNA as monomers in a head-to-tail manner. The neighboring Cren7 monomers are located on the opposite sides of the DNA duplex, with each introducing a single-step sharp kink by intercalation of the hydrophobic side chain of Leu28, bending the DNA into an S-shape conformation. A structural model for the chromatin fiber folded by Cren7 was established and verified by the analysis of cross-linked Cren7-DNA complexes by atomic force microscopy. Our results suggest that Cren7 differs significantly from Sul7, another chromatin protein conserved among Sulfolobus species, in both DNA binding and deformation. These data shed significant light on the strategy of chromosomal DNA organization in crenarchaea.

Comprehensive genotyping of the C9orf72 hexanucleotide repeat region in 2095 ALS samples from the NINDS collection using a two-mode, long-read PCR assay

Objective: Expansion of the G4C2 repeat tract in the C9orf72 gene is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Here, we provide comprehensive genotyping of the C9orf72 repeat region for the National Institute of Neurological Disorders and Stroke (NINDS) ALS collection (n = 2095), using a novel bimodal PCR assay capable of amplifying nearly 100% GC-rich sequences. Methods: A single-tube 3-primer PCR assay mode, resolved using capillary electrophoresis, was used for sizing up to 145 repeats with single-repeat accuracy, for detecting expansions irrespective of their overall size, and for flagging confounding 3′ sequence variations (SVs). A modified two-primer PCR mode, resolved via agarose gel electrophoresis, provided further size information for hyper-expanded samples (>145 repeats) up to ∼5.8 kb amplicons (∼950 G4C2 repeats). Results: Within the evaluated cohort, 177 (8.4%) samples were expanded, with 175 (99%) samples being hyper-expanded. 3′-SVs were identified in 64 (3.1%) samples, and were most common in expanded alleles. Genotypes of all 606 (29%) homozygous samples were confirmed using an orthogonal PCR assay. Conclusion: This study and PCR method may improve and standardize molecular characterization of the C9orf72 locus, and have the potential to inform phenotype–genotype correlations and therapeutic development in ALS/FTD.

The Role of GC-Rich Sequences from the Promoter Region of the Drosophila melanogaster yellow Gene in the Enhancer- Dependent Activation of Transcription.

It is shown that mutations in two GC-rich sequences (GC-boxes) from the promoter region of the yellow gene during enhancer-dependent transcription activation do not affect the basal level of the yellow gene transcription but destabilize the interaction between the enhancers and the promoter.

The role of GC-rich sequences from the promotor region of the Drosophila melanogaster yellow gene in the enhancer-dependent activation of transscription

The role of GC-rich sequences from the promotor region of the Drosophila melanogaster yellow gene in the enhancer-dependent activation of transscription

Protein Interactions in the T7 DNA Replisome Facilitate DNA Damage Bypass.

The DNA replisome inevitably encounters DNA damage during DNA replication. The T7 DNA replisome contains a DNA polymerase (gp5), the processivity factor thioredoxin (trx), a helicase-primase (gp4), and a ssDNA-binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated strand-displacement DNA synthesis past 8-oxoG or O6 -MeG lesions at the synthetic DNA fork by the T7 DNA replisome. DNA damage does not obviously affect the binding affinities between helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O6 -MeG-as well as GC-rich template sequence clusters-inhibit strand-displacement DNA synthesis and produce partial extension products. Relative to the gp4 ΔC-tail, gp4 promotes DNA damage bypass. The presence of gp2.5 also promotes it. Thus, the interactions of polymerase with helicase and ssDNA-binding protein facilitate DNA damage bypass. Accessory proteins in other complicated DNA replisomes also facilitate bypassing DNA damage in similar manner. This work provides new mechanistic information relating to DNA damage bypass by the DNA replisome.

Structural basis of chimpanzee APOBEC3H dimerization stabilized by double-stranded RNA.

APOBEC3H (A3H) is a mammal-specific cytidine deaminase that potently restricts the replication of retroviruses. Primate A3Hs are known to exert key selective pressures against the cross-species transmission of primate immunodeficiency viruses from chimpanzees to humans. Despite recent advances, the molecular structures underlying the functional mechanisms of primate A3Hs have not been fully understood. Here, we reveal the 2.20-Å crystal structure of the chimpanzee A3H (cpzA3H) dimer bound to a short double-stranded RNA (dsRNA), which appears to be similar to two recently reported structures of pig-tailed macaque A3H and human A3H. In the structure, the dsRNA-binding interface forms a specialized architecture with unique features. The analysis of the dsRNA nucleotides in the cpzA3H complex revealed the GC-rich palindrome-like sequence preference for dsRNA interaction, which is largely determined by arginine residues in loop 1. In cells, alterations of the cpzA3H residues critical for the dsRNA interaction severely reduce intracellular protein stability due to proteasomal degradation. This suggests that cpzA3H stability is regulated by the dsRNA-mediated dimerization as well as by unknown cellular machinery through proteasomal degradation in cells. Taken together, these findings highlight unique structural features of primate A3Hs that are important to further understand their cellular functions and regulation.