Double Base Substitutions Research Articles

Clinical Interest in Exome-Based Analysis of Somatic Mutational Signatures for Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality. This study investigates the clinical interest of whole exome sequencing (WES) for analyzing somatic mutational signatures in patients with advanced or metastatic NSCLC treated with the current standard of care. Exome sequencing data and clinical characteristics from 132 patients with advanced or metastatic NSCLC were analyzed. Somatic mutational signatures including single base substitutions (SBSs), double base substitutions (DBSs), and copy number signatures were evaluated. Structural variations including tumor mutational burden (TMB), the number of neoantigens, TCR clonality, homologous recombination deficiency (HRD), copy number alterations (CNAs), and microsatellite instability (MSI) score were determined. The association between these genomic features, NSCLC subtypes, and patient outcomes (progression-free and overall survival) was evaluated. Exome sequencing offers valuable insights into somatic mutational signatures in NSCLC. This study identified specific signatures associated with a poor response to immune checkpoint inhibitor (ICI) therapy and chemotherapy, potentially aiding treatment selection and identifying patients unlikely to benefit from these approaches.

High-efficiency genetic engineering toolkit for virus based on lambda red-mediated recombination.

Viruses, such as Ebola virus (EBOV), evolve rapidly and threaten the human health. There is a great demand to exploit efficient gene-editing techniques for the identification of virus to probe virulence mechanism for drug development. Based on lambda Red recombination in Escherichia coli (E. coli), counter-selection, and in vitro annealing, a high-efficiency genetic method was utilized here for precisely engineering viruses. EBOV trVLPs assay and dual luciferase reporter assay were used to further test the effect of mutations on virus replication. Considering the significance of matrix protein VP24 in EBOV replication, the types of mutations within vp24, including several single-base substitutions, one double-base substitution, two seamless deletions, and one targeted insertion, were generated on the multi-copy plasmid of E. coli. Further, the length of the homology arms for recombination and in vitro annealing, and the amount of DNA cassettes and linear plasmids were optimized to create a more elaborate and cost-efficient protocol than original approach. The effects of VP24 mutations on the expression of a reporter gene (luciferase) from the EBOV minigenome were determined, and results indicated that mutations of key sites within VP24 have significant impacts on EBOV replication. This precise mutagenesis method will facilitate effective and simple editing of viral genes in E. coli.

Abstract 6463: Persistence of smoking mutational signatures in the non-small cell lung cancer genome

Abstract Mutational signatures offer insights into cancer etiology. Smoking tobacco is associated with three signatures involving single base substitutions (SBS4), double base substitutions (DBS2) and insertions/deletions (ID3). Combining mutational signatures with clinical data allows examination of temporal and quantitative aspects of carcinogenic exposures. We analyzed 132 non-small cell lung carcinoma (NSCLC) whole genomes, sequenced through the 100,000 Genomes Project (Genomics England), with paired clinical data available for 130/132 (98.5%). Clinical data included smoking status, duration, pack-years (py), and duration of smoking cessation. The majority were “ever-smokers” (ex-smokers: 90/130, 69.2%; current smokers: 32/130, 24.6%) and 120/122 (98.4%) had at least one smoking signature. DBS2 was the most sensitive signature for ever-smokers (118/122, 96.7%), although SBS4 was the optimal signature for diagnosing smoking-related NSCLCs (SBS4 positive likelihood ratio 7.15). An absence of ID3 best identified true never smoker NSCLCs (ID3 negative likelihood ratio 0.08). Ever-smokers had a higher tumor mutational burden (TMB) than never smokers (median 8.25 mutations/Mb vs 1.18 mutations/Mb, p = <0.0001), while TMB positively correlated with smoking pack-years (p = 0.044). Following smoking cessation, the median length of discontinuation was 11 years before a smoking-related NSCLC was diagnosed and, in some cases, smoking signatures persisted for up to 50 years prior to NSCLC diagnosis. To complement existing epidemiological data, 40 pack years was estimated as a critical cut-off for minimizing ongoing NSCLC risk. Prolonged smoking cessation did not reduce TMB (p = 0.055) or SBS4 mutations (p = 0.11), although this did produce lower DBS2 counts (p = 0.0067). Further signatures observed included SBS92 (previously seen in the bladder urothelium of ever-smokers) and a novel signature hypothesized to be linked to DNA mismatch repair deficiencies. NSCLCs from two patients with prior platinum chemo-radiotherapy harbored signatures of both chemotherapy- and radiotherapy-induced damage in their recurrent biopsy sample. These results highlight the importance of paired sequencing and clinical data, revealing that smoking-induced DNA damage can have long-lived effects, and strengthening health guidance of abstinence from smoking at the earliest possibility. Citation Format: Colin R. Lindsay, Pantelis Nicola, Andreas Gruber, Kate Brown, Mathew Carter, Helen Adderley, Shereen Rafee, Anna Moss, Andrew Wallace, George Burghel, Helene Schlecht, Katie Baker, Sharzad Moghadam, Jane Rogan, Jamie Weaver, Angeliki Malliri, William Newman, Fiona Blackhall, David Wedge, Genomics England. Persistence of smoking mutational signatures in the non-small cell lung cancer genome. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6463.

The mutational impact of Illudin S on human cells

Illudin S (ILS) is a fungal sesquiterpene secondary metabolite with potent genotoxic and cytotoxic properties. Early genetic studies and more recent genome-wide CRISPR screens showed that Illudin-induced lesions are preferentially repaired by transcription-coupled nucleotide excision repair (TC-NER) with some contribution from post-replication repair pathways. In line with these results, Irofulven, a semi-synthetic ILS analog was recently shown to be particularly effective on cell lines and patient-derived xenografts with impaired NER (e.g. ERCC2/3 mutations), raising hope that ILS-derived molecules may soon enter the clinic. Despite the therapeutic potential of ILS and its analogs, we still lack a global understanding of their mutagenic potential. Here, we characterize the mutational signatures associated with chronic exposure to ILS in human cells. ILS treatment rapidly stalls DNA replication and transcription, leading to the activation of the replication stress response and the accumulation of DNA damage. Novel single and double base substitution signatures as well as a characteristic indel signature indicate that ILS treatment preferentially alkylates purine residues and induces oxidative stress, confirming prior in vitro data. Many mutation contexts exhibit a strong transcriptional strand bias, highlighting the contribution of TC-NER to the repair of ILS lesions. Finally, collateral mutations are also observed in response to ILS, suggesting a contribution of translesion synthesis pathways to ILS tolerance. Accordingly, ILS treatment led to the rapid recruitment of the Y-family DNA polymerase kappa onto chromatin, supporting its preferential use for ILS lesion bypass. Altogether, our work provides the first global assessment of the genomic impact of ILS, demonstrating the contribution of multiple DNA repair pathways to ILS resistance and mutagenicity.

Association of APOBEC3A/B deletion variant with endometrial cancer risk in Norwegian population: A case-control study (242)

<b>Objectives:</b> APOBEC3 family of proteins play important roles in intrinsic responses to viral infection by introducing mutations into the viral nucleic acids. APOBEC3 activity targeting the host cell's DNA may lead to mutations contributing to tumorigenesis. Uterine cancers are found to be linked with three APOBEC associated signatures; single-base substitution signatures 2 and -13 and double-base substitution signature 11. A germline deletion of 30Kb in chromosome 22 affecting the APOBEC3A and -B genes leads to the formation of a hybrid transcript, APOBEC3A/B, found to be more stable than the wild-type APOBEC3A and causes more DNA damage through higher protein levels. To the best of our knowledge, the potential association between the APOBEC3A/B deletion variant and the risk of endometrial cancer has not yet been assessed. This study aimed to evaluate the APOBEC3A/B deletion as a potential risk modulating factor for endometrial cancer. <b>Methods:</b> DNA extracted from blood samples of Norwegian (Caucasian) women diagnosed with primary uterine adenocarcinoma (<i>n</i>=1,470), and healthy controls (<i>n</i>=1,918) were screened for germline APOBEC3A/B deletion variant using quantitative polymerase chain reaction high-resolution melting (qPCR-HMR) curves. For technical validation, 35.4% of cases were genotyped for SNP rs12628403 (Chr 22:38962032), an SNP in proximity to the APOBEC3A/B-deletion start point that is found in strong linkage disequilibrium with the deletion allele. The potential difference in results from APOBEC3A/B deletion and rs12628403-analyses were considered negligible due to a very low recombination rate of 5.8x10-3. <b>Results:</b> Cases and controls were in Hardy-Weinberg equilibrium for genotype distribution (p=0.78 and p>0.4, with a MAF of 0.072 and 0.094, respectively). Our study cohort showed a reduced risk of endometrial cancer in dominant and allele models (OR: 0.75, 95 % CI: 0.62-0.91, p=0.003 and OR: 0.75, 95 % CI: 0.63-0.90, p=0.0002, respectively). In a subgroup analysis by histology, endometrioid endometrial cancer showed similar patterns (dominant model: OR: 0.64, 95% CI: 0.51-0.79, p=0.1x10-3 and allele model: OR: 0.64, 95% CI: 0.52-0.79, p=0.1x10-3). No clear risk association was observed in non-endometrioid endometrial cancer subtype. Among age subgroups generated with a 10-year cut-off, dominant and allele models yielded a significantly reduced cancer risk in individuals in the age range 50-59 (OR: 0.51, 95% CI: 0.33-0.78, p=0.002 and OR: 0.54, 95% CI: 0.35-0.81, p=0.002, respectively) and 60-69 (OR = 0.62, 95% CI=0.43-0.88, p=0.8x10-3 and OR=0.62, CI=0.44-0.88, p=0.005, respectively). <b>Conclusions:</b> APOBEC3A/B deletion variant is associated with reduced endometrial cancer risk in Norwegian women.

Abstract 3627: The genomic landscape of breast cancers from CHEK2 c.1100delC mutation carriers

Abstract Women who carry the germline CHEK2 c.1100delC mutation have a 2.3-fold increased life-time risk to develop breast cancer (BC). Their BCs are mostly of the luminal/ER+ subtype and diagnosed at a younger age than BCs in sporadic patients. BC patients carrying a CHEK2 c.1100delC mutation are at an increased risk of developing a contralateral BC and have a worse survival compared to sporadic BC patients although resistance to either endocrine or chemotherapy does not appear to play a role herein. To unravel the biological mechanism by which CHEK2 c.1100delC exerts its tumorigenic potential, whole genome sequencing was performed of matching normal and primary BC (pBC) DNA from twenty female CHEK2 c.1100delC mutation carriers at Hartwig Medical Foundation (HMF). We compared the somatic pBC genomes of these twenty patients to 560 pBC genomes from the International Cancer Genome Consortium including 30 BRCA1 and 25 BRCA2 mutation carriers and 329 sporadic ER+ pBC patients. The validation cohort consisted of 517 metastatic BC (mBC) genomes from the Center for Personalized Cancer Treatment that were sequenced at HMF and included 24 mBC genomes from CHEK2 c.1100delC mutation carriers. We found that CHEK2 pBC and mBC genomes were dissimilar to pBC and mBC genomes of BRCA1 and BRCA2 mutation carriers and did not show evidence of a homologous recombination deficiency (HRD) phenotype. Instead, CHEK2 pBC and mBC genomes were very similar to genomes of sporadic ER+ pBC and mBC patients in terms of tumor mutational burden, single and double base substitution signatures as well as small insertion/deletion signatures. The size distribution of structural variants (SVs), however, was different for CHEK2 pBCs and mBCs compared with ER+ as well as BRCA1 and BRCA2 pBCs and mBCs. Importantly, the CHEK2 c.1100delC mutation was associated with an increased frequency of larger (&amp;gt; 1Mb) deletions, inversions and tandem duplications (TDs) with peaks at specific sizes. Although CHEK2 mBC genomes more frequently harbored chromothripsis compared with other mBC subtypes, this did not explain the CHEK2-specific SV size distribution pattern. Moreover, increased chromothripsis in CHEK2 mBC genomes was not associated with a higher frequency of somatic TP53 mutations. In fact, we did not observe any somatic TP53 mutations among CHEK2 pBC and mBC genomes. This mutual exclusiveness suggests CHEK2-driven tumorigenesis could act through the TP53 signaling pathway. To conclude, CHEK2 BC genomes do not harbor somatic TP53 mutations and display a unique SV size distribution profile. Moreover, the absence of an HRD phenotype among CHEK2 BC genomes suggests PARP inhibitors will not be effective for treatment of BC in CHEK2 c.1100delC mutation carriers. Increasing knowledge of the mechanisms of CHEK2-driven tumorigenesis may ultimately lead to the development of tailored strategies for treatment and prevention of BC for CHEK2 mutation carriers. Citation Format: Marcel Smid, Marjanka K. Schmidt, Wendy J. Prager - van der Smissen, Kirsten Ruigrok - Ritstier, Sten Cornelissen, Maartje A. Schreurs, J. Margriet Collee, Muriel A. Adank, Maartje J. Hooning, John W. Martens, Antoinette Hollestelle. The genomic landscape of breast cancers from CHEK2 c.1100delC mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3627.

Comprehensive Molecular Profiling of Colorectal Cancer With Situs Inversus Totalis by Next-Generation Sequencing.

BackgroundColorectal cancer (CRC) is one of the most prevalent malignances worldwide. However, CRC with situs inversus totalis (SCRC) is extremely rare, and molecular characterization of this disease has never been investigated.MethodsTumor tissue samples from 8 patients with SCRC and 33 CRC patients without situs inversus totalis (NSCRC) were subjected to multigene next-generation sequencing.ResultsThe most frequently mutated genes in SCRC were APC, TP53, CHEK2, MDC1, GNAQ, KRAS, and SMAD4. A high frequency of SCRC tumors had mutations in DNA damage repair genes. Single amino acid substitutions in the DNA damage repair genes caused by continuous double base substitution was identified in the majority of this population. Furthermore, mutational profiles showed notable differences between the SCRC and NSCRC groups. In particular, CHEK2, MDC1, GNAQ, SMAD4, BRCA1, HLA-B, LATS2, and NLRC5 mutations were more frequently observed in SCRC patients. The mutation loci distributions of KRAS in the SCRC cohort differed from that of the NSCRC cohort. Additionally, differences in the targeted genomic profiles and base substitution patterns were observed between the two groups.ConclusionsThese findings comprehensively revealed a molecular characterization of SCRC, which will contribute to the development of personalized therapy and improved clinical management of SCRC patients.

SomaticSiMu: a mutational signature simulator.

SomaticSiMu is an in silico simulator of single and double base substitutions, and single base insertions and deletions in an input genomic sequence to mimic mutational signatures. SomaticSiMu outputs simulated DNA sequences and mutational catalogues with imposed mutational signatures. The tool is the first mutational signature simulator featuring a graphical user interface, control of mutation rates and built-in visualization tools of the simulated mutations. Simulated datasets are useful as a ground truth to test the accuracy and sensitivity of DNA sequence classification tools and mutational signature extraction tools under different experimental scenarios. The reliability of SomaticSiMu was affirmed by (i) supervised machine learning classification of simulated sequences with different mutation types and burdens, and (ii) mutational signature extraction from simulated mutational catalogues. SomaticSiMu is written in Python 3.8.3. The open-source code, documentation and tutorials are available at https://github.com/HillLab/SomaticSiMu under the terms of the CreativeCommonsAttribution4.0InternationalLicense. Supplementary data are available at Bioinformatics online.

MutationalPatterns: the one stop shop for the analysis of mutational processes

BackgroundThe collective of somatic mutations in a genome represents a record of mutational processes that have been operative in a cell. These processes can be investigated by extracting relevant mutational patterns from sequencing data.ResultsHere, we present the next version of MutationalPatterns, an R/Bioconductor package, which allows in-depth mutational analysis of catalogues of single and double base substitutions as well as small insertions and deletions. Major features of the package include the possibility to perform regional mutation spectra analyses and the possibility to detect strand asymmetry phenomena, such as lesion segregation. On top of this, the package also contains functions to determine how likely it is that a signature can cause damaging mutations (i.e., mutations that affect protein function). This updated package supports stricter signature refitting on known signatures in order to prevent overfitting. Using simulated mutation matrices containing varied signature contributions, we showed that reliable refitting can be achieved even when only 50 mutations are present per signature. Additionally, we incorporated bootstrapped signature refitting to assess the robustness of the signature analyses. Finally, we applied the package on genome mutation data of cell lines in which we deleted specific DNA repair processes and on large cancer datasets, to show how the package can be used to generate novel biological insights.ConclusionsThis novel version of MutationalPatterns allows for more comprehensive analyses and visualization of mutational patterns in order to study the underlying processes. Ultimately, in-depth mutational analyses may contribute to improved biological insights in mechanisms of mutation accumulation as well as aid cancer diagnostics. MutationalPatterns is freely available at http://bioconductor.org/packages/MutationalPatterns.

The dose-, LET-, and gene-dependent patterns of DNA changes underlying the point mutations in spermatozoa of Drosophila melanogaster. I. Autosomal gene black.

Sequence analysis of 7 spontaneous, 27 γ-ray- and 20 neutron/neutron+γ-ray-induced black (b) point mutants was carried out. All these mutants were isolated as non-mosaic transmissible recessive visibles in the progeny of irradiated males from the wild-type high-inbred laboratory D32 strain of Drosophila melanogaster. Among spontaneous mutants, there were two (28.5 %) mutants with copia insertion in intron 1 and exon 2, three (42.8 %) with replacement of b+D32 paternal sequence with maternal b1 sequence (gene conversion), one (14.3 %) with 142-bp-long insertion in exon 2, and one (14.3 %) with a short deletion and two single-base substitutions in exon 3. Among γ-ray-induced mutants, there were 1 (3.7 %) with copia insertion in intron 2, 6 (22.2 %) with gene conversion, and the remaining 20 (74.1 %) mutants had 37 different small-scale DNA changes. There were 20 (54.1 %) single- or double-base substitutions, 7 (18.9 %) frameshifts (indels), 9 (24.3 %) extended deletions or insertions, and 1(2.7 %) mutant with a short insertion instead of a short deletion. Remarkably, clusters of independent small-scale changes inside the gene or within one DNA helical turn were recovered. The spectrum of DNA changes in 20 neutron/ neutron+γ-ray-induced mutants was drastically different from that induced by γ-rays in that 18 (90.0 %) mutants had the b1sequence. In addition, 2 (10.0 %) with gene conversion had 600- or 19-bp-long deletion in exon 3 and 1 (5.0 %) mutant with a short insertion instead of a short deletion. Analysis of all 27 mutants with gene conversion events shows that 20 (74.1 %) had full b1 sequence whereas 7 others (25.9 %) contained a partial b1 sequence. These data are the first experimental evidence for gene conversion in the early stages of animal embryogenesis in the first diploid cleavage nucleus after male and female pronuclei have united. The gene conversion, frameshifts (indels), and deletions between short repeats were considered as products of a relevant DNA repair pathways described in the literature. As the first step, the gametic doubling doses for phenotypic black point mutations and for intragenic base substitution mutations in mature sperm cells irradiated by 40 Gy of γ-rays were estimated as 5.8 and 1.2 Gy, respectively, showing that doubling dose for mutations at the molecular level is about 5 times lower than that at the phenotypic level.

Polymorphism analysis of propeller domain of k13 gene in Plasmodium ovale curtisi and Plasmodium ovale wallikeri isolates original infection from Myanmar and Africa in Yunnan Province, China

BackgroundEighteen imported ovale malaria cases imported from Myanmar and various African countries have been reported in Yunnan Province, China from 2013 to 2018. All of them have been confirmed by morphological examination and 18S small subunit ribosomal RNA gene (18S rRNA) based PCR in YNRL. Nevertheless, the subtypes of Plasmodium ovale could not be identified based on 18S rRNA gene test, thus posing challenges on its accurate diagnosis. To help establish a more sensitive and specific method for the detection of P. ovale genes, this study performs sequence analysis on k13-propeller polymorphisms in P. ovale.MethodsDried blood spots (DBS) from ovale malaria cases were collected from January 2013 to December 2018, and the infection sources were confirmed according to epidemiological investigation. DNA was extracted, and the coding region (from 206th aa to 725th aa) in k13 gene propeller domain was amplified using nested PCR. Subsequently, the amplified products were sequenced and compared with reference sequence to obtain CDS. The haplotypes and mutation loci of the CDS were analysed, and the spatial structure of the amino acid peptide chain of k13 gene propeller domain was predicted by SWISS-MODEL.ResultsThe coding region from 224th aa to 725th aa of k13 gene from P. ovale in 83.3% of collected samples (15/18) were amplified. Three haplotypes were observed in 15 samples, and the values of Ka/Ks, nucleic acid diversity index (π) and expected heterozygosity (He) were 3.784, 0.0095, and 0.4250. Curtisi haplotype, Wallikeri haplotype, and mutant type accounted for 73.3% (11/15), 20.0% (3/15), and 6.7% (1/15). The predominant haplotypes of P. ovale curtisi were determined in all five Myanmar isolates. Of the ten African isolates, six were identified as P. o. curtisi, three were P. o. wallikeri and one was mutant type. Base substitutions between the sequences of P. o. curtisi and P. o. wallikeri were determined at 38 loci, such as c.711. Moreover, the A > T base substitution at c.1428 was a nonsynonymous mutation, resulting in amino acid variation of T476S in the 476th position. Compared with sequence of P. o. wallikeri, the double nonsynonymous mutations of G > A and A > T at the sites of c.1186 and c.1428 leads to the variations of D396N and T476S for the 396th and 476th amino acids positions. For P. o. curtisi and P. o. wallikeri, the peptide chains in the coding region from 224th aa to 725th aa of k13 gene merely formed a monomeric spatial model, whereas the double-variant peptide chains of D396N and T476S formed homodimeric spatial model.ConclusionThe propeller domain of k13 gene in the P. ovale isolates imported into Yunnan Province from Myanmar and Africa showed high differentiation. The sequences of Myanmar-imported isolates belong to P. o. curtisi, while the sequences of African isolates showed the sympatric distribution from P. o. curtisi, P. o. wallikeri and mutant isolates. The CDS with a double base substitution formed a dimeric spatial model to encode the peptide chain, which is completely different from the monomeric spatial structure to encode the peptide chain from P. o. curtisi and P. o. wallikeri.

Abstract IA04: Drug resistance in end-stage high-grade serous ovarian cancer

Abstract Background: Acquired drug resistance is the major obstacle in controlling high-grade serous ovarian cancer (HGSC) and leads to poor overall survival. Comparatively little massively parallel sequencing data exist from HGSC patients with recurrent or end-stage disease who have been extensively treated with chemotherapy or newer targeted agents, such as antiangiogenics or PARP inhibitors (PARPi). We previously identified four mechanisms of acquired resistance from whole-genome sequencing (WGS) of 23 HGSC patients with recurrent disease; a resistance mechanism of any kind was only identified in half of the patients. Aim: To characterize genomic features and identify mechanisms of acquired drug resistance in end-stage HGSC patients. Methods: Since 2012, we have collected tumor samples from 19 HGSC patients through our rapid-autopsy program. On average, we collect 17 tumor sites per patient during the autopsy, which is performed a median 6 hours after death. SNP array, WGS, and RNA sequencing were performed to detect germline and somatic mutations, copy number alterations and gene expression patterns. Results: A total of 89 end-stage tumor samples from 15 patients with germline or somatic mutations in components of the homologous recombination (HR) DNA repair pathway have been analyzed by SNP array, and a subset of those have undergone WGS (n=44) and RNAseq (n=47). As expected, all samples had a somatic pathogenic TP53 mutation and have highly aberrant somatic copy number profiles. Prominent mutational signatures (single- and double-base substitutions) are associated with defective HR and prior treatment with platinum drugs. The total single-base substitution mutation burden is similar across tumor samples within patients; however, many mutations are unique to individual tumor samples. Currently, reversion mutations, which are secondary mutations in HR genes, are the most common resistance mechanism identified in our cohort of end-stage patients. Conclusion: Our data are allowing us to understand HGSC at end stage, by examining the catalogue of resistance mechanisms within an individual patient and reconstructing the natural history of HGSC. Citation Format: Elizabeth L. Christie, Kathryn Alsop, George Au-Yeung, Ahwan Pandey, Australian Ovarian Cancer Study, David D.L. Bowtell. Drug resistance in end-stage high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA04.

DNA polymerase ζ generates tandem mutations in immunoglobulin variable regions

Low-fidelity DNA polymerases introduce nucleotide substitutions in immunoglobulin variable regions during somatic hypermutation. Although DNA polymerase (pol) η is the major low-fidelity polymerase, other DNA polymerases may also contribute. Existing data are contradictory as to whether pol ζ is involved. We reasoned that the presence of pol η may mask the contribution of pol ζ, and therefore we generated mice deficient for pol η and heterozygous for pol ζ. The frequency and spectra of hypermutation was unaltered between Polζ(+/-) Polη(-/-) and Polζ(+/+) Polη(-/-) clones. However, there was a decrease in tandem double-base substitutions in Polζ(+/-) Polη(-/-) cells compared with Polζ(+/+) Polη(-/-) cells, suggesting that pol ζ generates tandem mutations. Contiguous mutations are consistent with the biochemical property of pol ζ to extend a mismatch with a second mutation. The presence of this unique signature implies that pol ζ contributes to mutational synthesis in vivo. Additionally, data on tandem mutations from wild type, Polζ(+/-), Polζ(-/-), Ung(-/-), Msh2(-/-), Msh6(-/-), and Ung(-/-) Msh2(-/-) clones suggest that pol ζ may function in the MSH2-MSH6 pathway.

An intrastrand three-DNA-base interaction is a key specificity determinant of F transfer initiation and of F TraI relaxase DNA recognition and cleavage

Bacterial conjugation, transfer of a single conjugative plasmid strand between bacteria, diversifies prokaryotic genomes and disseminates antibiotic resistance genes. As a prerequisite for transfer, plasmid-encoded relaxases bind to and cleave the transferred plasmid strand with sequence specificity. The crystal structure of the F TraI relaxase domain with bound single-stranded DNA suggests binding specificity is partly determined by an intrastrand three-way base-pairing interaction. We showed previously that single substitutions for the three interacting bases could significantly reduce binding. Here we examine the effect of single and double base substitutions at these positions on plasmid mobilization. Many substitutions reduce transfer, although the detrimental effects of some substitutions can be partially overcome by substitutions at a second site. We measured the affinity of the F TraI relaxase domain for several DNA sequence variants. While reduced transfer generally correlates with reduced binding affinity, some oriT variants transfer with an efficiency different than expected from their binding affinities, indicating ssDNA binding and cleavage do not correlate absolutely. Oligonucleotide cleavage assay results suggest the essential function of the three-base interaction may be to position the scissile phosphate for cleavage, rather than to directly contribute to binding affinity.

Mutation spectrum in UVB‐exposed skin epidermis of Xpa‐knockout mice: Frequent recovery of triplet mutations

Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the lambda-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa(-/-)) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa(-/-) and wild-type mice. Neither photolesion was removed in the Xpa(-/-) epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m(2) UVB increased the lacZ mutant frequency in the epidermis of Xpa(-/-) mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa(-/-) mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa(-/-) and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C --> T transitions at dipyrimidine sites; however, Xpa(-/-) mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC --> TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds.

Frequent recovery of triplet mutations in UVB-exposed skin epidermis of Xpc-knockout mice

Mutations of the Xpc gene cause a deficiency in global genome repair, a subpathway of nucleotide excision repair (NER), in mammalian cells. We used transgenic mice harboring the λ-phage-based lacZ mutational reporter gene to study the effect of an Xpc null mutation ( Xpc −/− ) on damage induction, repair and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpc −/− and wild-type mice. CPDs were not significantly removed in either of the mouse genotypes by 12 h after irradiation, whereas removal of 64PPs was observed in the wild-type. Irradiation with 300 and 400 J/m 2 UVB increased the lacZ mutant frequency in the Xpc −/− epidermis to at least twice as high as in the wild-type. Ninety-nine lacZ mutants isolated from the UVB-exposed epidermis of Xpc −/− mice were analyzed and compared with mutant sequences from irradiated wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in the dominance of C → T transitions at dipyrimidine sites; however, Xpc −/− mice had a higher frequency of two-base tandem substitutions, including CC → TT mutations, three-base tandem substitutions and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We concluded that the triplet mutation is a UV-specific mutation that preferably occurs in NER deficient genetic backgrounds.

Sequence Dependence of Charge Transport through DNA Domains

Here we examine the photooxidation of two kinetically fast electron hole traps, N4-cyclopropylcytosine (CPC) and N2-cyclopropylamine-guanosine (CPG), incorporated in DNA duplexes of various sequence using different photooxidants. DNA oxidation studies are carried out either with noncovalently bound [Ru(phen)(dppz)(bpy')]3+ (dppz = dipyridophenazine) and [Rh(phi)2(bpy)]3+ (phi = phenanthrenequinone diimine) or with anthraquinone tethered to DNA. Because the cyclopropylamine-substituted bases decompose rapidly upon oxidation, their efficiency of decomposition provides a measure of relative hole localization. Consistent with a higher oxidation potential for CPC versus CPG in DNA, CPC decomposes with photooxidation by [Rh(phi)2(bpy)]3+, while CPG undergoes ring-opening both with photoexcited [Rh(phi)2(bpy)]3+ and with [Ru(phen)(dppz)(bpy')]3+. Anthraquinone-modified DNA assemblies of identical base composition but different base sequence are also probed. Single and double base substitutions within adenine tracts modulate CPC decomposition. In fact, the entire sequence within the DNA assembly is seen to govern CPC oxidation, not simply the bases intervening between CPC and the tethered photooxidant. These data are reconciled in the context of a mechanistic model of conformationally gated charge transport through delocalized DNA domains. Photooxidations of anthraquinone-modified DNA assemblies containing both CPC and CPG, but with varied distances separating the modified bases, point to a domain size of at least three bases. Our model for DNA charge transport is distinguished from polaron models. In our model, delocalized domains within the base pair stack form transiently based upon sequence-dependent DNA structure and dynamics. Given these results, DNA charge transport is indeed remarkably sensitive to DNA sequence and structure.

Direct detection of 16S rRna using oligonucleotide microarrays assisted by base stacking hybridization and tyramide signal amplification

A simple method has been developed and validated for direct, sensitive detection and specific identification of 16S rRNA. We first report our direct investigation of discrimination efficiency for sequence variations in RNA using oligonucleotide microarrays assisted by base stacking hybridization, and demonstrate that the sequence variations of double base substitution, single base substitution and single base deletion in RNA could be directly identified. With the help of tyramide signal amplification (TSA), the detection sensitivity of this method for four clinically important bacterial species was below 0.5, 5, 1 and 1 ng of total RNA, which are 100–1000 fold more sensitive than the published methods.

Direct detection of 16S rRna using oligonucleotide microarrays assisted by base stacking hybridization and tyramide signal amplification

A simple method has been developed and validated for direct, sensitive detection and specific identification of 16S rRNA. We first report our direct investigation of discrimination efficiency for sequence variations in RNA using oligonucleotide microarrays assisted by base stacking hybridization, and demonstrate that the sequence variations of double base substitution, single base substitution and single base deletion in RNA could be directly identified. With the help of tyramide signal amplification (TSA), the detection sensitivity of this method for four clinically important bacterial species was below 0.5, 5, 1 and 1 ng of total RNA, which are 100–1000 fold more sensitive than the published methods.

Mutational analysis of the bglH catabolite-responsive element (cre) in Lactobacillus plantarum.

Catabolite repression of the Lactobacillus plantarum bglH gene is mediated by the catabolite control protein A (CcpA). Here we show that the binding site for the protein is a catabolite-responsive element (cre) sequence overlapping the start site of transcription. Two single and one double base substitutions in the cre sequence caused derepression of a plasmid-borne bglH-cat transcriptional fusion in L. plantarum cells grown on glucose. Analysis of the single mutations indicates that the G and C nucleotide residues in positions 2 and 13, respectively, of the 14-bp cre sequence are required for catabolite repression.