A chemical method to sequence 5-formylcytosine on RNA

Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method to sequence 5-formylcytosine (f5C) modifications on RNA based upon selective and efficient malononitrile-mediated labeling of f5C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and PCR amplification. We apply Mal-Seq to characterize the prevalence of f5C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f5C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f5C epitranscriptome.

The effect of flanking bases on direct and triplet sensitized cyclobutane pyrimidine dimer formation in DNA depends on the dipyrimidine, wavelength and the photosensitizer

Cyclobutane pyrimidine dimers (CPDs) are the major products of DNA produced by direct absorption of UV light, and result in C to T mutations linked to human skin cancers. Most recently a new pathway to CPDs in melanocytes has been discovered that has been proposed to arise from a chemisensitized pathway involving a triplet sensitizer that increases mutagenesis by increasing the percentage of C-containing CPDs. To investigate how triplet sensitization may differ from direct UV irradiation, CPD formation was quantified in a 129-mer DNA designed to contain all 64 possible NYYN sequences. CPD formation with UVB light varied about 2-fold between dipyrimidines and 12-fold with flanking sequence and was most frequent at YYYR and least frequent for GYYN sites in accord with a charge transfer quenching mechanism. In contrast, photosensitized CPD formation greatly favored TT over C-containing sites, more so for norfloxacin (NFX) than acetone, in accord with their differing triplet energies. While the sequence dependence for photosensitized TT CPD formation was similar to UVB light, there were significant differences, especially between NFX and acetone that could be largely explained by the ability of NFX to intercalate into DNA.

Comparison of cytosine base editors and development of the BEable-GPS database for targeting pathogenic SNVs

A variety of base editors have been developed to achieve C-to-T editing in different genomic contexts. Here, we compare a panel of five base editors on their C-to-T editing efficiencies and product purity at commonly editable sites, including some human pathogenic C-to-T mutations. We further profile the accessibilities of 20 base editors to all possible pathogenic mutations in silico. Finally, we build the BEable-GPS (Base Editable prediction of Global Pathogenic SNVs) database for users to select proper base editors to model or correct disease-related mutations. The in vivo comparison and in silico profiling catalog the availability of base editors and their broad applications in biomedical studies.

A direct multi-generational estimate of the human mutation rate from autozygous segments seen in thousands of parentally related individuals

Heterozygous mutations within homozygous sequences descended from a recent common ancestor offer a way to ascertain de novo mutations (DNMs) across multiple generations. Using exome sequences from 3,222 British-Pakistani individuals with high parental relatedness, we estimate a mutation rate of 1. 45 ± 0.05 × 10−8 per base pair per generation in autosomal coding sequence, with a corresponding noncrossover gene conversion rate of 8.75 ± 0.05 × 10−6 per base pair per generation. This is at the lower end of exome mutation rates previously estimated in parent-offspring trios, suggesting that post-zygotic mutations contribute little to the human germline mutation rate. We found frequent recurrence of mutations at polymorphic CpG sites, and an increase in C to T mutations in a 5’ CCG 3’ → 5’ CTG 3’ context in the Pakistani population compared to Europeans, suggesting that mutational processes have evolved rapidly between human populations.

Strand-biased cytosine deamination at the replication fork causes cytosine to thymine mutations inEscherichia coli

The rate of cytosine deamination is much higher in single-stranded DNA (ssDNA) than in double-stranded DNA, and copying the resulting uracils causes C to T mutations. To study this phenomenon, the catalytic domain of APOBEC3G (A3G-CTD), an ssDNA-specific cytosine deaminase, was expressed in anEscherichia colistrain defective in uracil repair (ungmutant), and the mutations that accumulated over thousands of generations were determined by whole-genome sequencing. C:G to T:A transitions dominated, with significantly more cytosines mutated to thymine in the lagging-strand template (LGST) than in the leading-strand template (LDST). This strand bias was present in both repair-defective and repair-proficient cells and was strongest and highly significant in cells expressing A3G-CTD. These results show that the LGST is accessible to cellular cytosine deaminating agents, explains the well-known GC skew in microbial genomes, and suggests the APOBEC3 family of mutators may target the LGST in the human genome.

Analysis of BCR-ABL1 Tyrosine Kinase Domain Mutations In Primitive Chronic Myeloid Leukemia Cells Identifies a Unique Mutator Phenotype.

Abstract 3397 The tyrosine kinase inhibitor (TKI), imatinib mesylate (IM), induces remissions in most chronic phase chronic myeloid leukaemia (CML-CP) patients, but emergence of drug resistance attributable to critical mutations in the BCR-ABL1 TK domain remains a significant clinical problem. We previously demonstrated that primitive (CD34+) CML-CP cells are both intrinsically insensitive to IM and genetically unstable. BCR-ABL1 TK domain mutations are, furthermore, readily detectable in CD34+ CML-CP cells even prior to IM treatment. However, the mechanisms by which they arise are unknown. In this study, we characterized the spectrum of mutations at each of the 3 codon positions in the BCR-ABL1 TK domain of IM-naïve CD34+ cells and used a mathematical model to compare these with those found in the same region of DNA in IM-resistant cells. A total of 460 TK mutations were identified in cells from 15 IM-naïve and from 316 IM-resistant patients. The mutations were non-randomly distributed across the 3 codon positions, with very few mutations at position 3 (1 of 136 mutations, 0.7%, in IM-naïve patients and 31 of 329, 9%, in IM-resistant patients). The remainder showed no bias in their distribution across codon positions 1 and 2 in IM-naïve patient cells but, in IM-resistant cells, a significant positive bias was observed at codon position 2 (p=0.042). Conversely, BCR-ABL1 TK domain sequences from IM-naïve patient cells had significantly more transitions relative to unselected regions of the genome across all codon positions, a significant positive A-to-G mutational bias (A>G, 1.12×10−10) and an under-representation of C-to-T mutations (C>T, 5.34×10−5) when these mutations were compared with unselected regions of the genome. In addition, we observed a T-to-C mutational hotspot (T>C, 1.23×10−4) at codon position 2. IM-naïve cells exhibited a transitional bias at position 2 and overall (1.99×10−4 and 6.19×10−5, respectively; at position 1, P=0.052). We observed a similar pattern of TK codon position mutations at position 1 and overall in sequences derived from IM-resistant cells. Interestingly, in IM-resistant cells, A-to-T (A>T) transversions were over-represented at position 2 and overall (1.41×10−11 and 3.77×10−5). These cells also had a profound transversional bias (1.45×10−4) at codon position 3, suggesting that positive selection occurs at a position where transversions are typically non-synonymous. However, a skewed transition:transversion ratio and transition and transversion frequency at TK codon positions 1 and 2 was observed in both IM-naïve and IM-selected cells with mutations distributed across these 2 codon positions in a significantly uneven fashion. In IM-resistant cells, the frequency of C-to-T mutations (C>T, 3.82×10−11) indicated a bias affecting codon position 2 more often than position 1, and A-to-T transversions occurred significantly more frequently at position 2 compared to position 1 (A>T, 7.45×10−9). This was also the case for T-to-C transitions in TK sequences of IM-naïve cells (T>C, 1.13×10−4). Frequencies of these TK mutations in IM-naïve and IM-resistant cells were also higher than in unselected regions of the genome, providing further evidence that the TK domain mutations in CML-CP cells have a distinct mutational profile. For example, the clinically observed M244V and D276G mutations result from A-to-G transitions and the F359L mutation arises from a T-to-C transition, both of which would be predicted by the activity of a CML-CP mutator. However, the most clinically important mutation, T315I, that confers resistance to most currently available TKIs, is generated by a C-to-T transition, suggesting a high mutational rate generating mutational escape around the principal mutator pattern and profound selection thereafter. Notably, the CML-CP mutational signature is distinct from that of the activation-induced cytidine deaminase (AID)-induced hypermutation reported in CML blast crisis, both in targeting specific codon positions and in the overall mutational pattern. This strongly favors the hypothesis that the genomic instability of primitive CML-CP cells is generated by a different, specific mutational process. The results also suggest that monitoring BCR-ABL mutational hotspots may be clinically useful in anticipating TKI resistance. Disclosures: No relevant conflicts of interest to declare.

Transcription-Dependent Increase in Multiple Classes of Base Substitution Mutations in Escherichia coli

ABSTRACT We showed previously that transcription in Escherichia coli promotes C · G-to-T · A transitions due to increased deamination of cytosines to uracils in the nontranscribed but not the transcribed strand (A. Beletskii and A. S. Bhagwat, Proc. Natl. Acad. Sci. USA 93:13919-13924, 1996). To study mutations other than that of C to T, we developed a new genetic assay that selects only base substitution mutations and additionally excludes C · G to T · A transitions. This novel genetic reversion system is based on mutations in a termination codon and involves positive selection for resistance to bleomycin or kanamycin. Using this genetic system, we show here that transcription from a strong promoter increases the level of non-C-to-T as well as C-to-T mutations. We find that high-level transcription increases the level of non-C-to-T mutations in DNA repair-proficient cells in three different sequence contexts in two genes and that the rate of mutation is higher by a factor of 2 to 4 under these conditions. These increases are not caused by a growth advantage for the revertants and are restricted to genes that are induced for transcription. In particular, high levels of transcription do not create a general mutator phenotype in E. coli. Sequence analysis of the revertants revealed that the frequency of several different base substitutions increased upon transcription of the bleomycin resistance gene and that G · C-to-T · A transversions dominated the spectrum in cells transcribing the gene. These results suggest that high levels of transcription promote many different spontaneous base substitutions in E. coli.

Transcription-Induced Cytosine-to-Thymine Mutations Are Not Dependent on Sequence Context of the Target Cytosine

ABSTRACT We showed previously that transcription of a plasmid-bornekan allele increases C-to-T mutations in the nontranscribed strand. Using two new plasmid-borne kanalleles, one cmp allele, and a chromosomalkan allele, we found in this study that transcription-induced mutations are not limited to specific genes, alleles, or locations and are likely to be a general property of transcript elongation in Escherichia coli.

Action of Repeat-Induced Point Mutation on Both Strands of a Duplex and on Tandem Duplications of Various Sizes in Neurospora

In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (repeat-induced point mutation). Affected sequences are subjected to multiple GC-to-AT mutations. To explore the pattern in which base changes are laid down by RIP we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event at the mtr locus. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication flanking the zeta-eta (ζ-η) locus. The mtr sequence data were also used to further examine the basis for the differential severity of C-to-T mutations on the coding and noncoding strands in genes. The known bias of RIP toward CpA/TpG sites in conjunction with the sequence bias of Neurospora accounts for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) within the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications is consistent with the idea that reversion of long tandem duplications and RIP share a common step.