Non-transcribed Strand Research Articles

Dynamics of transcription-coupled repair of cyclobutane pyrimidine dimers and (6-4) photoproducts in Escherichia coli

DNA repair processes modulate genotoxicity, mutagenesis, and adaption. Nucleotide excision repair removes bulky DNA damage, and in Escherichia coli, basal excision repair, carried out by UvrA, B, C, and D, with DNA PolI and DNA ligase, occurs genome-wide. In transcription-coupled repair (TCR), the Mfd protein targets template strand (TS) lesions that block RNA polymerase for accelerated repair by the basal repair enzymes. Accelerated repair is also seen with particular adducts. Notably, of the two major UV photoproducts, basal repair of (6-4) photoproducts [(6-4)PPs] is about 10× faster than repair of cyclobutane pyrimidine dimers (CPDs). To better understand repair prioritization in E. coli, we used XR-seq to measure TCR of UV photoproducts genome-wide. With CPDs, we found that TCR occurred at early time points, increased with transcription level, and was Mfd dependent; later, with completion of TS repair, nontranscribed strand (NTS) repair predominated. With (6-4)PP, when analyzing all genes, TCR was not observed; in fact, among the most highly transcribed genes, slightly more repair of (6-4)PPs in the NTS was evident. Thus, the very rapid basal repair of (6-4)PP in the NTS was faster than TCR of (6-4)PPs in the TS. Overall, TCR is of limited importance in (6-4)PP repair, and TCR of CPDs is limited to the TS of more highly transcribed genes. These results are consistent with the significant role of Mfd in mutagenesis and the modest effect of mfd deletion on UV survival and bear upon the response of E. coli to bulky DNA damage.

Mutations of the circadian clock genes Cry, Per, or Bmal1 have different effects on the transcribed and nontranscribed strands of cycling genes

One important goal of circadian medicine is to apply time-of-day dosing to improve the efficacy of chemotherapy. However, limited knowledge of how the circadian clock regulates DNA repair presents a challenge to mechanism-based clinical application. We studied time-series genome-wide nucleotide excision repair in liver and kidney of wild type and three different clock mutant genotypes (Cry1-/-Cry2-/-, Per1-/-Per2-/-, and Bmal1-/-). Rhythmic repair on the nontranscribed strand was lost in all three clock mutants. Conversely, rhythmic repair of hundreds of genes on the transcribed strand (TSs) persisted in the livers of Cry1-/-Cry2-/- and Per1-/-Per2-/- mice. We identified a tissue-specific, promoter element-driven repair mode on TSs of collagen and angiogenesis genes in the absence of clock activators or repressors. Furthermore, repair on TSs of thousands of genes was altered when the circadian clock is disrupted. These data contribute to a better understanding of the regulatory role of the circadian clock on nucleotide excision repair in mammals and may be invaluable toward the design of time-aware platinum-based interventions in cancer.

Assessment of the roles of Spt5-nucleic acid contacts in promoter proximal pausing of RNA polymerase II

Promoter proximal pausing of RNA polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires the transcription factor DRB sensitivity-inducing factor (DSIF) and the inhibitory negative elongation factor (NELF). DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We used a highly purified invitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. We identified two domains of Spt5, the KOW4 and NGN domains, that facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short helical motif that is in close proximity to the non-transcribed DNA template strand. Removal of this sequence in Drosophila has a male-specific dominant negative effect. The alpha-helical motif is also needed to support fly viability. We also show that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex. Disruption of the KOW1-DNA interaction is dominant lethal invivo. Finally, we show that the KOW2-3 domain of Spt5 mediates the recruitment of NELF to the elongation complex. In summary, our results reveal additional roles for DSIF in transcription regulation and identify specific domains important for facilitating Pol II pausing.

Molecular Mechanisms of Transcription-Coupled Repair.

Transcription-coupled repair (TCR), discovered as preferential nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers located in transcribed mammalian genes compared to those in nontranscribed regions of the genome, is defined as faster repair of the transcribed strand versus the nontranscribed strand in transcribed genes. The phenomenon, universal in model organisms including Escherichia coli, yeast, Arabidopsis, mice, and humans, involves a translocase that interacts with both RNA polymerase stalled at damage in the transcribed strand and nucleotide excision repair proteins to accelerate repair. Drosophila, a notable exception, exhibits TCR but lacks an obvious TCR translocase. Mutations inactivating TCR genes cause increased damage-induced mutagenesis in E. coli and severe neurological and UV sensitivity syndromes in humans. To date, only E. coli TCR has been reconstituted in vitro with purified proteins. Detailed investigations of TCR using genome-wide next-generation sequencing methods, cryo-electron microscopy, single-molecule analysis, and other approaches have revealed fascinating mechanisms.

Spontaneous deamination of cytosine to uracil is biased to the non-transcribed DNA strand in yeast

Transcription in Saccharomyces cerevisiae is associated with elevated mutation and this partially reflects enhanced damage of the corresponding DNA. Spontaneous deamination of cytosine to uracil leads to CG>TA mutations that provide a strand-specific read-out of damage in strains that lack the ability to remove uracil from DNA. Using the CAN1 forward mutation reporter, we found that C>T and G>A mutations, which reflect deamination of the non-transcribed and transcribed DNA strands, respectively, occurred at similar rates under low-transcription conditions. By contrast, the rate of C>T mutations was 3-fold higher than G>A mutations under high-transcription conditions, demonstrating biased deamination of the non-transcribed strand (NTS). The NTS is transiently single-stranded within the ∼15 bp transcription bubble, or a more extensive region of the NTS can be exposed as part of an R-loop that can form behind RNA polymerase. Neither the deletion of genes whose products restrain R-loop formation nor the over-expression of RNase H1, which degrades R-loops, reduced the biased deamination of the NTS, and no transcription-associated R-loop formation at CAN1 was detected. These results suggest that the NTS within the transcription bubble is a target for spontaneous deamination and likely other types of DNA damage.

The Mfd protein is the transcription-repair coupling factor (TRCF) in Mycobacterium smegmatis

Invitro and invivo experiments with Escherichia coli have shown that the Mfd translocase is responsible for transcription-coupled repair, a subpathway of nucleotide excision repair involving the faster rate of repair of the transcribed strand than the nontranscribed strand. Even though the mfd gene is conserved in all bacterial lineages, there is only limited information on whether it performs the same function in other bacterial species. Here, by genome scale analysis of repair of UV-induced cyclobutane pyrimidine dimers, we find that the Mfd protein is the transcription-repair coupling factor in Mycobacterium smegmatis. This finding, combined with the inverted strandedness of UV-induced mutations in WT and mfd-E.coli and Bacillus subtilis indicate that the Mfd protein is the universal transcription-repair coupling factor in bacteria.

Nucleotide excision repair removes thymidine analog 5-ethynyl-2′-deoxyuridine from the mammalian genome

Nucleotide excision repair is the principal mechanism for removing bulky DNA adducts from the mammalian genome, including those induced by environmental carcinogens such as UV radiation, and anticancer drugs such as cisplatin. Surprisingly, we found that the widely used thymidine analog EdU is a substrate for excision repair when incorporated into the DNA of replicating cells. A number of thymidine analogs were tested, and only EdU was a substrate for excision repair. EdU excision was absent in repair-deficient cells, and invitro, DNA duplexes bearing EdU were also substrates for excision by mammalian cell-free extracts. We used the excision repair sequencing (XR-seq) method to map EdU repair in the human genome at single-nucleotide resolution and observed that EdU was excised throughout the genome and was subject to transcription-coupled repair as evidenced by higher repair rates in the transcribed strand (TS) relative to the nontranscribed strand (NTS) in transcriptionally active genes. These properties of EdU, combined with its cellular toxicity and ability to cross the blood-brain barrier, make it a potential candidate for treating cancers of the brain, a tissue that typically demonstrates limited replication in adults.

Aristolochic acid-containing Chinese herbal medicine and upper urinary tract urothelial carcinoma in Taiwan: a narrative review.

The high incidence of upper urinary tract urothelial carcinoma (UTUC) in Taiwan is largely due to exposure to aristolochic acid (AA), a principal component of Aristolochia-based herbal medicines. Here we systematically review the molecular epidemiology, clinical presentation and biomarkers associated with AA-induced UTUC. This is a narrative review. Medline, Embase, and Web of Science were searched from inception to December 31, 2021. Studies evaluating the association, detection, and clinical characteristics of AA and UTUC were included. A nationwide database revealed 39% of the Taiwanese population had been exposed to AA-containing herbs between 1997 and 2003. Epidemiological reports revealed AA posed a significantly higher hazard for renal failure and UTUC in herbalists and the general population who ingested AA-containing herbs. The presence of aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, located predominantly on the non-transcribed DNA strand, with a strong preference for deoxyadenosine in a consensus sequence (CAG), was observed in many UTUC patients. Clinically, AA-related UTUC patients were characterized by a younger age, female gender, impaired renal function and recurrence of contralateral UTUC. To date, there are no preventive measures, except prophylactic nephrectomy, for subjects at risk of AA nephropathy or AA-related UTUC. AA exposure via Aristolochia-based herbal medicines is a problem throughout Taiwan, resulting in a high incidence of UTUC. Aristolactam-DNA adducts and a distinctive signature mutation, A:T to T:A transversions, can be used as biomarkers to identify AA-related UTUC. AA-related UTUC is associated with a high recurrence rate of contralateral UTUC.

The landscape of genetic alterations of UVB-induced skin tumors in DNA repair-deficient mice.

Non-melanoma skin cancer (NMSC) is mainly caused by ultraviolet (UV)-induced somatic mutations and is characterized by UV signature modifications. Xeroderma pigmentosum group A (Xpa) knockout mice exhibit extreme UV-induced photo-skin carcinogenesis, along with a photosensitive phenotype. We performed whole-exome sequencing (WES) of squamous cell carcinoma (SCC) samples after repetitive ultraviolet B (UVB) exposure to investigate the differences in the landscape of somatic mutations between Xpa knockout and wild-type mice. Although the tumors that developed in mice harboured UV signature mutations in a similar set of cancer-related genes, the pattern of transcriptional strand asymmetry was largely different; UV signature mutations in Xpa knockout and wild-type mice preferentially occurred in transcribed and non-transcribed strands, respectively, reflecting a deficiency in transcription-coupled nucleotide excision repair in Xpa knockout mice. Serial time point analyses of WES for a tumor induced by only a single UVB exposure showed pathogenic mutations in Kras, Fat1, and Kmt2c, which may be driver genes for the initiation and promotion of SCC in Xpa knockout mice. Furthermore, the inhibitory effects on tumor production in Xpa knockout mice by the anti-inflammatory CXCL1 monoclonal antibody affected the pattern of somatic mutations, wherein the transcriptional strand asymmetry was attenuated and the activated signal transduction was shifted from the RAS/RAF/MAPK to the PIK3CA pathway.

Transcription of ncRNAs promotes repair of UV induced DNA lesions in Saccharomyces cerevisiae subtelomeres.

Ultraviolet light causes DNA lesions that are removed by nucleotide excision repair (NER). The efficiency of NER is conditional to transcription and chromatin structure. UV induced photoproducts are repaired faster in the gene transcribed strands than in the non-transcribed strands or in transcriptionally inactive regions of the genome. This specificity of NER is known as transcription-coupled repair (TCR). The discovery of pervasive non-coding RNA transcription (ncRNA) advocates for ubiquitous contribution of TCR to the repair of UV photoproducts, beyond the repair of active gene-transcribed strands. Chromatin rules transcription, and telomeres form a complex structure of proteins that silences nearby engineered ectopic genes. The essential protective function of telomeres also includes preventing unwanted repair of double-strand breaks. Thus, telomeres were thought to be transcriptionally inert, but more recently, ncRNA transcription was found to initiate in subtelomeric regions. On the other hand, induced DNA lesions like the UV photoproducts must be recognized and repaired also at the ends of chromosomes. In this study, repair of UV induced DNA lesions was analyzed in the subtelomeric regions of budding yeast. The T4-endonuclease V nicking-activity at cyclobutene pyrimidine dimer (CPD) sites was exploited to monitor CPD formation and repair. The presence of two photoproducts, CPDs and pyrimidine (6,4)-pyrimidones (6-4PPs), was verified by the effective and precise blockage of Taq DNA polymerase at these sites. The results indicate that UV photoproducts in silenced heterochromatin are slowly repaired, but that ncRNA transcription enhances NER throughout one subtelomeric element, called Y', and in distinct short segments of the second, more conserved element, called X. Therefore, ncRNA-transcription dependent TCR assists global genome repair to remove CPDs and 6-4PPs from subtelomeric DNA.

Set2 histone methyltransferase regulates transcription coupled-nucleotide excision repair in yeast.

Helix-distorting DNA lesions, including ultraviolet (UV) light-induced damage, are repaired by the global genomic-nucleotide excision repair (GG-NER) and transcription coupled-nucleotide excision repair (TC-NER) pathways. Previous studies have shown that histone post-translational modifications (PTMs) such as histone acetylation and methylation can promote GG-NER in chromatin. Whether histone PTMs also regulate the repair of DNA lesions by the TC-NER pathway in transcribed DNA is unknown. Here, we report that histone H3 K36 methylation (H3K36me) by the Set2 histone methyltransferase in yeast regulates TC-NER. Mutations in Set2 or H3K36 result in UV sensitivity that is epistatic with Rad26, the primary TC-NER factor in yeast, and cause a defect in the repair of UV damage across the yeast genome. We further show that mutations in Set2 or H3K36 in a GG-NER deficient strain (i.e., rad16Δ) partially rescue its UV sensitivity. Our data indicate that deletion of SET2 rescues UV sensitivity in a GG-NER deficient strain by activating cryptic antisense transcription, so that the non-transcribed strand (NTS) of yeast genes is repaired by TC-NER. These findings indicate that Set2 methylation of H3K36 establishes transcriptional asymmetry in repair by promoting canonical TC-NER of the transcribed strand (TS) and suppressing cryptic TC-NER of the NTS.

Spectroscopic Characterization of Mitochondrial G-Quadruplexes.

Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies.

The mRNA tether model for activation-induced deaminase and its relevance for Ig somatic hypermutation and class switch recombination

Activation-induced deaminase (AID) only deaminates cytosine within single-stranded DNA. Transcription is known to increase AID deamination on duplex DNA substrates during transcription. Using a purified T7 RNA polymerase transcription system, we recently found that AID deamination of a duplex DNA substrate is reduced if RNase A is added during transcription. This finding prompted us to consider that the mRNA tail may contribute to AID action at the nearby transcribed strand (TS) or non-transcribed strand (NTS) of DNA, which are transiently single-stranded in the wake of RNA polymerase movement. Here, we used a purified system to test whether a single-stranded oligonucleotide (oligo) consisting of RNA in the 5′ portion and DNA in the 3′ portion (i.e., 5′RNA-DNA3′, also termed an RNA-DNA fusion substrate) could be deaminated equally efficiently as the same sequence when it is entirely DNA. We found that AID acts on the RNA-DNA fusion substrate and the DNA-only substrate with similar efficiency. Based on this finding and our recent observation on the importance of the mRNA tail, we propose a model in which the proximity and length of the mRNA tail provide a critical site for AID loading to permit a high local collision frequency with the NTS and TS in the transient wake of the RNA polymerase. When the mRNA tail is not present, we know that AID action drops to levels equivalent to when there is no transcription at all. This mRNA tether model explains several local and global features of Ig somatic hypermutation and Ig class switch recombination, while integrating structural and functional features of AID.

The Efficiency of Global Genome-Nucleotide Excision Repair is Linked to the Fraction of Open rRNA Gene Chromatin, in Yeast.

The yeast rDNA locus is a suitable model to study nucleotide excision repair (NER) in chromatin. A portion of rRNA genes is transcribed and largely depleted of nucleosomes, the remaining genes are not transcribed and folded in nucleosomes. In G1-arrested cells, most rRNA genes do not have nucleosomes. TC-NER removes UV-induced DNA lesions from the transcribed strand of active genes. GG-NER is less efficient and removes DNA lesions from the nontranscribed strand of active genes and from the inactive genome. Different from mammalian cells, in yeast, the rRNA gene-transcribed strand is repaired by RNA polymerase-I-dependent TC-NER. The opposite nontranscribed strand is repaired faster than both strands of inactive rRNA genes. In log-phase cells, RNA polymerase-I are dislodged from the damaged transcribed strand and partially replaced by nucleosomes. Contrary to log-phase cells, in G1-phase cells few, if any, histones are deposited on the open rRNA genes during NER. In this study, we compared GG-NER efficiency in the rRNA gene coding region: without nucleosomes, partially loaded or wholly loaded with nucleosomes. The results indicate that in log-phase cells histones obstruct GG-NER, whereas in G1-phase cells GG-NER is as efficient as TC-NER.

Tandem Substitutions in Somatic Hypermutation

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. While tandem substitutions have been described in mice and other species, the incidence of this phenomenon and its underlying mechanism in humans is currently unknown.We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors generated by unbiased ARTISAN PCR. Selection of unique, clonally unrelated, antigen-experienced sequences carrying up to 5% mutations yielded 13.532 VDJ, 7.952 VJ-kappa and 7.598 VJ-lambda. Comparison to the closest germline allele allowed for identification of a total of 122.878 single nucleotide substitutions (SNS), 10.735 tandem dinucleotide substitutions (TDNS) and 2.615 longer contiguous substitutions. After correcting for expected clusters of adjacent SNS, tandem substitutions comprised 5,7% of all AID-induced mutations. The mutation of more than one nucleotide in a single event, was shown to overcome amino acid codon redundancy and may therefore enhance the adaptive immune response. Clustering of such mutations around AID hotspots and their overall distribution indicates that tandem substitutions are an integral part of the SHM spectrum.In the majority of tandem substitutions, the mutated sequence may be identified in the directly adjacent reference sequence context. Tandem substitutions in humans therefore represent single nucleotide juxtalocations. Such juxtalocations appear to be favored in polydipyramidine stretches. These observations could be confirmed in patients with MSH2/6 deficiency, but were absent in a VDJ library from an UNG-deficient patient, indicating a strict dependence on abasic sites as an instigating mechanism.Together, these findings delineate a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions.Our study shows that a significant portion of mutations acquired during SHM are caused by tandem substitutions, and that this mechanism may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously.Figure legend. Corrected incidence of tandem dinucleotide substitutions in healthy donors. (A) Dinucleotide substitutions from unique IGHV, IGKV and IGLV sequences and corrected after in silico predictions of dinucleotide substitutions that did not occur in tandem. Burgundy cells represent sequence inversions, light and dark purple cells represent juxtalocations of the 5‘ and 3‘ base in the pair (as seen from the non-transcribed strand), respectively. For unshaded cells, juxtalocation could not be assessed due to one or more nucleotides in the reference sequence matching the mutated sequence. (B) Relative contribution of sequence inversions and juxtalocations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Selection and thermostability suggest G-quadruplexes are novel functional elements of the human genome.

Approximately 1% of the human genome has the ability to fold into G-quadruplexes (G4s)—noncanonical strand-specific DNA structures forming at G-rich motifs. G4s regulate several key cellular processes (e.g., transcription) and have been hypothesized to participate in others (e.g., firing of replication origins). Moreover, G4s differ in their thermostability, and this may affect their function. Yet, G4s may also hinder replication, transcription, and translation and may increase genome instability and mutation rates. Therefore, depending on their genomic location, thermostability, and functionality, G4 loci might evolve under different selective pressures, which has never been investigated. Here we conducted the first genome-wide analysis of G4 distribution, thermostability, and selection. We found an overrepresentation, high thermostability, and purifying selection for G4s within genic components in which they are expected to be functional—promoters, CpG islands, and 5′ and 3′ UTRs. A similar pattern was observed for G4s within replication origins, enhancers, eQTLs, and TAD boundary regions, strongly suggesting their functionality. In contrast, G4s on the nontranscribed strand of exons were underrepresented, were unstable, and evolved neutrally. In general, G4s on the nontranscribed strand of genic components had lower density and were less stable than those on the transcribed strand, suggesting that the former are avoided at the RNA level. Across the genome, purifying selection was stronger at stable G4s. Our results suggest that purifying selection preserves the sequences of functional G4s, whereas nonfunctional G4s are too costly to be tolerated in the genome. Thus, G4s are emerging as fundamental, functional genomic elements.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

Exons and introns exhibit transcriptional strand asymmetry of dinucleotide distribution, damage formation and DNA repair.

Recent cancer sequencing efforts have uncovered asymmetry in DNA damage induced mutagenesis between the transcribed and non-transcribed strands of genes. Here, we investigate the major type of damage induced by ultraviolet (UV) radiation, the cyclobutane pyrimidine dimers (CPDs), which are formed primarily in TT dinucleotides. We reveal that a transcriptional asymmetry already exists at the level of TT dinucleotide frequency and therefore also in CPD damage formation. This asymmetry is conserved in vertebrates and invertebrates and is completely reversed between introns and exons. We show the asymmetry in introns is linked to the transcription process itself, and is also found in enhancer elements. In contrast, the asymmetry in exons is not correlated to transcription, and is associated with codon usage preferences. Reanalysis of nucleotide excision repair, normalizing repair to the underlying TT frequencies, we show repair of CPDs is more efficient in exons compared to introns, contributing to the maintenance and integrity of coding regions. Our results highlight the importance of considering the primary sequence of the DNA in determining DNA damage sensitivity and mutagenic potential.

Presumptive Evidence for ADAR1 A-to-I Deamination at WA-sites as the Mutagenic Genomic Driver in Hepatocellular and Related ADAR1-Hi Cancers

In hepatocellular cancer (HCC) there is an over expression of the RNA editing enzyme ADAR1. Further, the prominent genomic somatic mutation signature in HCC is almost exclusively focused on mutations at A:T base pairs where A-to-G mutations far exceed T-to-C mutations (when read on the non-transcribed strand). A clear mechanism for this extreme transcriptional strand biased mutation signature, putatively associated with over expression of ADAR1 deaminase, is yet to be explicitly demonstrated. The standard description of this strand bias has been nominally called “Transcription Coupled Damage” (TCD) to distinguish it from more conventional “Transcription Coupled Repair” (TCR). We show that the TCD description does not satisfy all features of the molecular evidence. The conventional view is that ADAR1 is thought to target adenosines at WA-sites for editing to inosine (I) in double stranded RNA stem-loop structures in transcripts. Here we show that the totality of the molecular and cellular data on these mutation signatures provides strong presumptive evidence for a clear role for ADAR1-mediated A-to-I deamination at WA-sites as the mutagenic driver in hepatocellular and possibly other related ADAR1-Hi cancers displaying biased mutation features at A:T base pairs.

CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene.

Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation.