Replication Errors Research Articles

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine.

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves ‘on-the-fly’ translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for lesion bypass. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.

Mismatch-Repair Protein Expression in High-Grade Gliomas: A Large Retrospective Multicenter Study.

Background: DNA mismatch repair (MMR) is a system for repairing errors in DNA replication. Cancer cells with MMR deficiency can have immunohistochemical loss of MMR protein expression leading to a hypermutable phenotype that may correlate with anti-PD1 efficacy. Scant data exist about immunohistochemical loss of MMR protein expression in high-grade gliomas (HGG). Materials and Methods: We performed a large multicenter retrospective study to investigate the frequency and the prognostic role of immunohistochemical loss of MMR protein expression in HGG patients; we nevertheless evaluated the association between this status and clinical or molecular characteristics. Immunohistochemical loss of MMR protein expression was recorded as partial or complete loss of at least 1 MMR protein. Results: We analyzed the expression of MMR proteins in tumor tissue of 355 consecutive patients. Partial and complete immunohistochemical loss of MMR proteins was found in 43/355 samples (12.1%) and among these, 15 cases (4.2%) showed a complete loss of at the least one MMR protein. Alteration of MSH2 expression was found in 55.8%, MSH6 in 46.5%, PMS2 in 34.9%, and MLH1 in 30.2%. Alteration of MMR protein expression was statistically more frequent in anaplastic gliomas, in recurrent disease, in patients treated with temozolomide, and in IDH-mut gliomas. Immunohistochemical loss of MMR proteins was not associated with survival, adjusting for clinically relevant confounders. Conclusions: MMR protein expression status did not affect survival in HGG patients. We identified clinical and molecular characteristics correlating with immunohistochemical loss of MMR proteins expression. A large study should be performed to analyze its predictive role of immune checkpoint inhibitor efficacy in these subgroups of patients.

Unique Structural Features of Mammalian NEIL2 DNA Glycosylase Prime Its Activity for Diverse DNA Substrates and Environments

Oxidative damage on DNA arising from both endogenous and exogenous sources can result in base modifications that promote errors in replication as well as generating sites of base loss (abasic sites) that present unique challenges to maintaining genomic integrity. These lesions are excised by DNA glycosylases in the first step of the base excision repair pathway. Here we present the first crystal structure of a NEIL2 glycosylase, an enzyme active on cytosine oxidation products and abasic sites. The structure reveals an unusual "open" conformation not seen in NEIL1 or NEIL3 orthologs. NEIL2 is predicted to adopt a "closed" conformation when bound to its substrate. Combined crystallographic and solution-scattering studies show the enzyme to be conformationally dynamic in a manner distinct among the NEIL glycosylases and provide insight into the unique substrate preference of this enzyme. In addition, we characterized three cancer variants of human NEIL2, namely S140N, G230W, and G303R.

Reduction of the Hedging Error in the Trinomial Tree Model

The research problem is how large the replication error is if an investor Delta hedges an European option over many short periods. In the project, the trinomial stock price tree is firstly built to simulate stock price changes. Then, Monte-Carlo approximation is used to put many short-period trinomial tree models together and derive the average hedging errors. The hedging errors become significantly smaller as periods involved in the trinomial tree models pass a certain number. This method can be used to create portfolios with European options.

Single-Cell Sequencing of Mouse Thymocytes Reveals Mutational Landscape Shaped by Replication Errors, Mismatch Repair, and H3K36me3.

SummaryDNA mismatch repair (MMR) corrects replication errors and is recruited by the histone mark H3K36me3, enriched in exons of transcriptionally active genes. To dissect in vivo the mutational landscape shaped by these processes, we employed single-cell exome sequencing on T cells of wild-type and MMR-deficient (Mlh1−/−) mice. Within active genes, we uncovered a spatial bias in MMR efficiency: 3′ exons, often H3K36me3-enriched, acquire significantly fewer MMR-dependent mutations compared with 5′ exons. Huwe1 and Mcm7 genes, both active during lymphocyte development, stood out as mutational hotspots in MMR-deficient cells, demonstrating their intrinsic vulnerability to replication error in this cell type. Both genes are H3K36me3-enriched, which can explain MMR-mediated elimination of replication errors in wild-type cells. Thus, H3K36me3 can boost MMR in transcriptionally active regions, both locally and globally. This offers an attractive concept of thrifty MMR targeting, where critical genes in each cell type enjoy preferential shielding against de novo mutations.

Abstract 5363: A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA

Abstract There is increasing interest in sequence analysis of circulating tumor cells (CTCs) to identify actionable mutations that may complement circulating tumor DNA (ctDNA) findings. Because the amount of DNA present in a single cell is miniscule (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as replication errors that can lead to false positive results. Targeted sequencing is an alternative approach that does not require WGA. We modified the CleanPlex® OncoZoom® Cancer Hotspot Panel protocol to perform targeted sequencing of single CTCs isolated by the RareCyte platform using A549 cell line spike-in and patient samples. We also used the CleanPlex panel to evaluate ctDNA from plasma isolated by blood collection tubes from two manufacturers (RareCyte and Streck). In both cases we compared OncoZoom results with and without prior WGA. Increased uniformity of coverage with decreased target dropout was observed when CleanPlex NGS libraries were prepared directly from cell lysates without WGA. Median read depth increased 48-fold when compared to the WGA method. On average, 23 out of 29 (79%) variants present in bulk A549 genomic DNA were observed without WGA in single model CTCs, while 15 of 29 (52%) were observed after WGA. Additionally, the false positive error frequency of non-WGA samples was 8-fold lower than in the WGA samples. CTCs and cell-free DNA from two metastatic breast cancer patients were also sequenced using OncoZoom. In one patient, PIK3CA E542K, a well-documented oncogenic mutation, was observed in 3 of 5 CTCs. In the other, ERBB2 L755S, a known HER2-reactivating mutation associated with chemoresistance, was found in 3 of 5 CTCs. Both patients' variants were present at similar allelic frequencies in plasma isolated from RareCyte and Streck cell-free DNA BCT tubes. Single-cell CleanPlex amplicon-based sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. This approach shows promise for cell-based liquid biopsy diagnostic applications. Citation Format: Nolan G. Ericson, Vidushi Kapoor, Guoying Liu, Alisa C. Clein, Daniel E. Sabath, Eric P. Kaldjian, Tad George. A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5363.

Abstract 2500: Copy number variation in recurrent BRCA1/2 germline mutation-associated breast and ovarian cancers

Abstract Carriers of pathogenic germline mutations in BRCA1/2 are highly predisposed to breast and ovarian cancers. Although their primary tumors respond to DNA-damaging agents, such as platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPi), these malignancies often return as therapy-resistant recurrences. To identify genetic mechanisms of therapeutic resistance, we performed whole exome sequencing on 41 pairs of matched primary/recurrent breast and ovarian tumors from 27 BRCA1/2 mutation carriers. The cohort consisted of 14 ovarian cancer patients (nine BRCA1, five BRCA2) and 13 breast cancer patients (nine BRCA1, four BRCA2). Five patients received PARPi, 17 patients received platinums, and all patients received some type of chemotherapy prior to recurrence. First, we performed a segmentation analysis to assess copy number variation (CNVs) in each tumor. We calculated homologous recombination deficiency (HRD) scores for each tumor, but found no significant differences between matched primary/recurrent pairs. We noted that primary tumors had a relatively high average HRD score (55/100), which remained high in recurrences (52/100). Next, we totaled arm-level CNVs to generate aneuploidy scores for each tumor. Using a Wilcoxon signed rank test, we found that aneuploidy scores were significantly higher in recurrences than in matched primary tumors (p=0.007). This finding suggests that recurrences have more arm-level CNVs than primary tumors, but that acquired genomic abnormalities are not caused by HRD per se. Instead, arm-level CNVs indicate that replication errors or genome doubling events are more common in recurrences than in primary tumors. Lastly, we annotated all tumors' CNVs with genes to interrogate potential effects on signaling pathways, including CNVs exclusive to each recurrence. Using Gene Set Enrichment Analysis with Hallmark gene sets, we identified pathways significantly (FDR q<0.30) affected by losses (CN≤1) and gains (CN≥4) from primary tumor CNVs, recurrent tumor CNVs, and recurrence-exclusive CNVs. All three CNV sets showed gains encompassing interferon gamma response genes and losses of Hedgehog signaling genes. Primary tumor losses were enriched for reactive oxygen species and cholesterol homeostasis genes. Recurrent tumor and recurrence-exclusive gains were enriched for UV-responsive and MTORC1 signaling gene sets, which may enhance survival in the presence of DNA-damaging or cytotoxic chemotherapies. These findings suggest that CNVs encompass different genes in recurrences compared to matched primary tumors. Further, differences in gene dosage and downstream signaling could represent novel therapeutic targets for recurrent tumors. Ultimately, we found that CNVs grant insights into the genomic and signaling processes that underlie acquired therapeutic resistance in BRCA1/2 mutation-associated cancers. Citation Format: Jennifer Brady Shah, Bradley Wubbenhorst, John Pluta, Caitlin Feltcher, Lauren Schmucker, Kurt D'Andrea, Heather Symecko, Catherine Ruan, Anupma Nayak, Kara N. Maxwell, Susan Domchek, Katherine L. Nathanson. Copy number variation in recurrent BRCA1/2 germline mutation-associated breast and ovarian cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2500.

Abstract 3537: Evaluation of genomic instability using microsatellite instability, ploidy status, and p53 expression in archival paraffin embedded tissue from primary ovarian cancer specimens

Abstract A retrospective study of a subset of ovarian cancer cases was evaluated to assess the role of microsatellite instability (MSI) and ploidy status. P53 function is important in the regulation of genomic stability and its expression was examined by immuno-histochemical staining. Genomic instability is also considered a possible indicator of immuno-therapy responsive tumors. Ovarian cancer patients diagnosed between 1983 to 1987 at Saskatoon Cancer Center, Canada, were evaluated for genomic stability. Nearly 400 Epithelial Ovarian Cancer (EOC) cases were reviewed, and Paraffin Embedded Tissue (PET) blocks from 112 cases were retrieved from hospitals in the region. PET blocks containing over 70% neoplastic tissue were chosen for the study. P53 expression was determined in eighteen cases using the mouse monoclonal antibody D07. An assessment of MSI and LOH was performed using the PCR mixes from Perkin Elmer (PE) AB. The replication error repair (RER)/Loss of Heterozygosity (LOH) assays were performed at nine loci using the primers obtained from PE. Fourteen out of eighteen cases were evaluated for ploidy status using a method described in literature[1]. Statistical analyses were performed to determine the significance of MSI/LOH, ploidy status, and p53 expression. In this study, 34 (30%) of 112 EOC cases were found to be aneuploid. Fourteen EOC cases had ploidy status that could be evaluated. Of the eighteen patients analyzed for MSI, at least nine patients showed instability at one or more loci. Of these nine, there were two RER positive phenotypes with aneuploid DNA content which were also p53 immuno-negative. Of the seven (78%) RER negative tumors, 57% (4/7) p53 negative, and the remaining three cases overexpressed p53. In five EOC cases, with an RER negative phenotype, 60% (3/5) were aneuploid and 40% (2/5) were diploid. Furthermore, LOH was a common event at the nine loci. The three events: MSI, lack of p53 overexpression, and aneuploidy, may be characteristics of a subset of ovarian cancer cases. Secondly, the study suggests that RER positivity occurs in ovarian cancer pathogenesis. We conclude that the data indicates general genomic instability due to MSI and DNA ploidy status. [1] A. Geissel and J. L. Griffin, “Preparation of nuclei for flow cytometry,” in AFIP Advances in Laboratory Methods in histology and Pathology, ed. Mikel UV (Washington DC: American Registry of Pathology, 1994), 111-121. Citation Format: Kavitha Advikolanu Rao, Anthony M. Magliocco, Andrew W. Maksymiuk. Evaluation of genomic instability using microsatellite instability, ploidy status, and p53 expression in archival paraffin embedded tissue from primary ovarian cancer specimens [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3537.

Abstract 740: Improved microsatellite instability detection in endometrial and prostate cancers using long mononucleotide repeat markers

Abstract Microsatellite instability (MSI) assays are used to screen cancer patients with mismatch repair deficiency (dMMR) for immune checkpoint inhibitor therapy. However, short mononucleotide repeat (SMR) markers in current MSI assays may sometimes display subtle shifts that are difficult to interpret in certain dMMR cancers, particular non-colorectal cancers. Long mononucleotide repeat (LMR) markers that are more prone to replication errors may improve the sensitivity of MSI assays. In this study, we compared the performance of SMR and LMR markers using two panels: (1) MSI Analysis System Version 1.2 (Promega) consisting of five SMR markers (21-27 bases in length) and (2) MSI LMR System (Promega) consisting of four SMR markers from the Version 1.2 panel and four LMR markers (52-60 bases in length). We studied 20 MMR proficient (pMMR) and 18 dMMR colorectal cancers (CRC) defined by concordant immunohistochemical staining (IHC) and the Version 1.2 MSI panel, 12 pMMR and 9 dMMR endometrial cancers (EC) defined by IHC alone, and 9 dMMR prostate cancers (PC) defined by loss of MSH2 IHC staining, loss of function MSH2 mutations, and increased mutation frequency. A marker was defined as unstable if a shift of 2 or more bases was observed in the tumor sample when compared to the normal sample. When using the Version 1.2 panel, we defined MSI high (MSI-H) as 2 or more unstable markers, MSI low (MSI-L) as 1 unstable marker, and microsatellite stable (MSS) as no shifts. With the LMR panel, we defined MSI-H as 3 or more unstable markers, MSI-L as 1 or 2 unstable markers, and MSS as no shifts. In dMMR CRC, the clinical sensitivity of the LMR and Version 1.2 panels for MSI were both 100%. In pMMR CRC, MSI-H was not observed in any cases using either panel, but MSI-L was observed in 2 and 0 cases using the LMR and Version 1.2 panels, respectively. In dMMR CRC, the average size of allelic shifts in the four LMR and SMR markers from the LMR panel were 8.7 and 22.1 bases, respectively. Germline polymorphisms were observed in 47% and 1% of LMR and SMR markers, respectively. In dMMR EC, the LMR panel showed greater sensitivity than the Version 1.2 panel (100% vs 89%). In pMMR EC, MSI-H was not observed in any cases using either panel, but MSI-L was observed more often using the LMR panel when compared to the Version 1.2 panel (2 vs 0 cases). In addition, LMR markers displayed larger shifts than SMR markers (12.1 vs 4.9 bases). Polymorphisms were also more frequent in LMR markers relative to SMR markers (60% vs 2%). Likewise, in dMMR PC, the sensitivity of the LMR panel was greater than that of the Version 1.2 panel (89% vs 44%). Furthermore, shifts in LMR markers were larger than those in SMR markers (10.3 vs 3.1 bases), and polymorphisms in LMR markers were more frequent than in SMR markers (33% vs 0%). Overall, LMR markers display greater clinical sensitivity and larger shifts, indicating that LMR markers can improve MSI detection in non-colorectal cancer. Citation Format: John H. Lin, Suping Chen, Liana B. Guedes, Emmanuel S. Antonarakis, Colin C. Pritchard, Tamara L. Lotan, James R. Eshleman. Improved microsatellite instability detection in endometrial and prostate cancers using long mononucleotide repeat markers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 740.

Mutational signature SBS8 predominantly arises due to late replication errors in cancer

Although a majority of somatic mutations in cancer are passengers, their mutational signatures provide mechanistic insights into mutagenesis and DNA repair processes. Mutational signature SBS8 is common in most cancers, but its etiology is debated. Incorporating genomic, epigenomic, and cellular process features for multiple cell-types we develop genome-wide composite epigenomic context-maps relevant for mutagenesis and DNA repair. Analyzing somatic mutation data from multiple cancer types in their epigenomic contexts, we show that SBS8 preferentially occurs in gene-poor, lamina-proximal, late replicating heterochromatin domains. While SBS8 is uncommon among mutations in non-malignant tissues, in tumor genomes its proportions increase with replication timing and speed, and checkpoint defects further promote this signature - suggesting that SBS8 probably arises due to uncorrected late replication errors during cancer progression. Our observations offer a potential reconciliation among different perspectives in the debate about the etiology of SBS8 and its relationship with other mutational signatures.

Evolution of a Human-Specific Tandem Repeat Associated with ALS

Tandem repeats are proposed to contribute to human-specific traits, and more than 40 tandem repeat expansions are known to cause neurological disease. Here, we characterize a human-specific 69bp variable number tandem repeat (VNTR) in the last intron of WDR7, which exhibits striking variability in both copy number and nucleotide composition, as revealed by long-read sequencing. In addition, greater repeat copy number is significantly enriched in three independent cohorts of individuals with sporadic amyotrophic lateral sclerosis (ALS). Each unit of the repeat forms a stem-loop structure with the potential to produce microRNAs, and the repeat RNA can aggregate when expressed in cells. We leveraged its remarkable sequence variability to align the repeat in 288 samples and uncover its mechanism of expansion. We found that the repeat expands in the 3'-5' direction, in groups of repeat units divisible by two. The expansion patterns we observed were consistent with duplication events, and a replication error called template switching. We also observed that the VNTR is expanded in both Denisovan and Neanderthal genomes but is fixed at one copy or fewer in non-human primates. Evaluating the repeat in 1000 Genomes Project samples reveals that some repeat segments are solely present or absent in certain geographic populations. The large size of the repeat unit in this VNTR, along with our multiplexed sequencing strategy, provides an unprecedented opportunity to study mechanisms of repeat expansion, and a framework for evaluating the roles of VNTRs in human evolution and disease.

Spontaneous Polyploids and Antimutators Compete During the Evolution of Saccharomyces cerevisiae Mutator Cells.

Mutations affecting DNA polymerase exonuclease domains or mismatch repair (MMR) generate "mutator" phenotypes capable of driving tumorigenesis. Cancers with both defects exhibit an explosive increase in mutation burden that appears to reach a threshold, consistent with selection acting against further mutation accumulation. In Saccharomyces cerevisiae haploid yeast, simultaneous defects in polymerase proofreading and MMR select for "antimutator" mutants that suppress the mutator phenotype. We report here that spontaneous polyploids also escape this "error-induced extinction" and routinely outcompete antimutators in evolved haploid cultures. We performed similar experiments to explore how diploid yeast adapt to the mutator phenotype. We first evolved cells with homozygous mutations affecting polymerase δ proofreading and MMR, which we anticipated would favor tetraploid emergence. While tetraploids arose with a low frequency, in most cultures, a single antimutator clone rose to prominence carrying biallelic mutations affecting the polymerase mutator alleles. Variation in mutation rate between subclones from the same culture suggests that there exists continued selection pressure for additional antimutator alleles. We then evolved diploid yeast modeling MMR-deficient cancers with the most common heterozygous exonuclease domain mutation (POLE-P286R). Although these cells grew robustly, within 120 generations, all subclones carried truncating or nonsynonymous mutations in the POLE-P286R homologous allele (pol2-P301R) that suppressed the mutator phenotype as much as 100-fold. Independent adaptive events in the same culture were common. Our findings suggest that analogous tumor cell populations may adapt to the threat of extinction by polyclonal mutations that neutralize the POLE mutator allele and preserve intratumoral genetic diversity for future adaptation.

Radioresistance, DNA Damage and DNA Repair in Cells With Moderate Overexpression of RPA1.

Molecular responses to genotoxic stress, such as ionizing radiation, are intricately complex and involve hundreds of genes. Whether targeted overexpression of an endogenous gene can enhance resistance to ionizing radiation remains to be explored. In the present study we take an advantage of the CRISPR/dCas9 technology to moderately overexpress the RPA1 gene that encodes a key functional subunit of the replication protein A (RPA). RPA is a highly conserved heterotrimeric single-stranded DNA-binding protein complex involved in DNA replication, recombination, and repair. Dysfunction of RPA1 is detrimental for cells and organisms and can lead to diminished resistance to many stress factors. We demonstrate that HEK293T cells overexpressing RPA1 exhibit enhanced resistance to cell killing by gamma-radiation. Using the alkali comet assay, we show a remarkable acceleration of DNA breaks rejoining after gamma-irradiation in RPA1 overexpressing cells. However, the spontaneous rate of DNA damage was also higher in the presence of RPA1 overexpression, suggesting alterations in the processing of replication errors due to elevated activity of the RPA protein. Additionally, the analysis of the distributions of cells with different levels of DNA damage showed a link between the RPA1 overexpression and the kinetics of DNA repair within differentially damaged cell subpopulations. Our results provide knew knowledge on DNA damage stress responses and indicate that the concept of enhancing radioresistance by targeted alteration of the expression of a single gene is feasible, however undesired consequences should be considered and evaluated.

Emergence and diversification of a host-parasite RNA ecosystem through Darwinian evolution.

In prebiotic evolution, molecular self-replicators are considered to develop into diverse, complex living organisms. The appearance of parasitic replicators is believed inevitable in this process. However, the role of parasitic replicators in prebiotic evolution remains elusive. Here, we demonstrated experimental coevolution of RNA self-replicators (host RNAs) and emerging parasitic replicators (parasitic RNAs) using an RNA-protein replication system we developed. During a long-term replication experiment, a clonal population of the host RNA turned into an evolving host-parasite ecosystem through the continuous emergence of new types of host and parasitic RNAs produced by replication errors. The host and parasitic RNAs diversified into at least two and three different lineages, respectively, and they exhibited evolutionary arms-race dynamics. The parasitic RNA accumulated unique mutations, thus adding a new genetic variation to the whole replicator ensemble. These results provide the first experimental evidence that the coevolutionary interplay between host-parasite molecules plays a key role in generating diversity and complexity in prebiotic molecular evolution.

Pricing and Hedging American-Style Options with Deep Learning

In this paper we introduce a deep learning method for pricing and hedging American-style options. It first computes a candidate optimal stopping policy. From there it derives a lower bound for the price. Then it calculates an upper bound, a point estimate and confidence intervals. Finally, it constructs an approximate dynamic hedging strategy. We test the approach on different specifications of a Bermudan max-call option. In all cases it produces highly accurate prices and dynamic hedging strategies with small replication errors.

The Sins of Our Forefathers: Paternal Impacts on De Novo Mutation Rate and Development.

Spermatogonial stem cells (SSCs) are generally characterized by excellent DNA surveillance and repair, resulting in one of the lowest spontaneous mutation rates in the body. However, the barriers to mutagenesis can be overwhelmed under two sets of circumstances. First, replication errors may generate age-dependent mutations that provide the mutant cells with a selective advantage, leading to the clonal expansions responsible for dominant genetic diseases such as Apert syndrome and achondroplasia. The second mechanism centers on the vulnerability of the male germline to oxidative stress and the induction of oxidative DNA damage in spermatozoa. Defective repair of such oxidative damage in the fertilized oocyte results in the creation of mutations in the zygote that can influence the health and well-being of the offspring. A particular hot spot for such oxidative attack on chromosome 15 has been found to align with several mutations responsible for paternally mediated disease, including cancer, psychiatric disorders, and infertility.

Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization

To investigate the role of oxidative stress-induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light-induced mutations in the cII gene from λ phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5' end of the mutated nucleotide. This mutation type was also enriched in the treated pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors because of oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of photosensitization-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells provide insights into the role of oxidative stress in senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair-defective human tumors.

Dynamic human MutSα–MutLα complexes compact mismatched DNA

DNA mismatch repair (MMR) corrects errors that occur during DNA replication. In humans, mutations in the proteins MutSα and MutLα that initiate MMR cause Lynch syndrome, the most common hereditary cancer. MutSα surveilles the DNA, and upon recognition of a replication error it undergoes adenosine triphosphate-dependent conformational changes and recruits MutLα. Subsequently, proliferating cell nuclear antigen (PCNA) activates MutLα to nick the error-containing strand to allow excision and resynthesis. The structure-function properties of these obligate MutSα-MutLα complexes remain mostly unexplored in higher eukaryotes, and models are predominately based on studies of prokaryotic proteins. Here, we utilize atomic force microscopy (AFM) coupled with other methods to reveal time- and concentration-dependent stoichiometries and conformations of assembling human MutSα-MutLα-DNA complexes. We find that they assemble into multimeric complexes comprising three to eight proteins around a mismatch on DNA. On the timescale of a few minutes, these complexes rearrange, folding and compacting the DNA. These observations contrast with dominant models of MMR initiation that envision diffusive MutS-MutL complexes that move away from the mismatch. Our results suggest MutSα localizes MutLα near the mismatch and promotes DNA configurations that could enhance MMR efficiency by facilitating MutLα nicking the DNA at multiple sites around the mismatch. In addition, such complexes may also protect the mismatch region from nucleosome reassembly until repair occurs, and they could potentially remodel adjacent nucleosomes.

Identification of Variants in Mitochondrial D-Loop and OriL Region and Analysis of Mitochondrial DNA Copy Number in Women with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a multifactorial disorder characterized by irregular menstrual problems, hyperandrogenism, and presence of polycystic ovaries. Till date, molecular mechanism underlying PCOS remains elusive. Recently mitochondrial displacement loop (D-loop) variants have been identified to be novel players in the pathogenesis of PCOS. At present, rare variants, besides common variants, are also the focus of research as it is believed to make essential contribution to the risk of complex diseases. However, rare and low hetroplasmic variants in mitochondrial D-loop are still not investigated in PCOS women. Furthermore, variants in light-strand origin of DNA replication (OriL) of mitochondrial DNA (mtDNA) have not been explored in PCOS. Hence, in this study, we investigated rare to common mitochondrial D-loop and OriL region variants obtained using mtDNA next-generation sequencing in women with PCOS. Furthermore, we also assessed mtDNA copy number, a biomarker of mitochondrial dysfunction (MD) in women with PCOS, as the variants in mtDNA are known to be associated with low mtDNA copy number in PCOS women. A total of 67 D-loop variants including 6 novel variants were identified in 30 PCOS women. Among 67 variants, 29 variants were reported in PCOS women. A single variant, 5746A was found in OriL region in two PCOS women. Both transition and transversion variants were found but transition variants occur at very high frequency compared with transversions (82.35% vs. 17.64%, respectively). As transition variants in mtDNA are known to arise because of polymerase γ errors, occurrence of high transition rates indicates that most mutation arises because of defect in replication errors that causes mtDNA damage leading to MD. Furthermore, mtDNA copy number was found to be low in women with PCOS compared with healthy control women suggesting that MD may be the contributing factor in the pathogenesis of PCOS.

Abstract PR13: Polymerase-mediated ultramutagenesis: A new approach for modeling the high mutational load of human cancer

Abstract Cancer is characterized by increased mutation rate, and recent work has led to a deeper understanding of mutator phenotypes and underlying molecular mechanisms. Differences in the mutational landscape of individual cancers underlie key aspects of clinical behavior. For example, overall base substitution rate is the best predictor of immune therapy response. Most genetically engineered mouse models of cancer (GEMMs) reiterate a few driver events but fail to recapitulate the high mutational loads that are the ultimate cause of most human cancer, making GEMMs inadequate for many aspects of tumor behavior, such as immune responses. The highest base substitution rates in cancers (≥100/Mb) result from specific heterozygous single amino acid substitutions (usually P286R) in the proofreading domain of DNA polymerase epsilon (POLE), rendering POLE highly error prone. POLE mutations are most common in endometrial cancer but occur with lower incidence in a wide range of cancers. We hypothesized that GEMMs could be generated by recapitulating PoleP286R via conditional knockin. Previously, we showed that constitutive expression of PoleP286R throughout the body triggered malignancies of diverse lineages. Mice harboring LSL-PoleP286R and the endometrial BAC-Sprr2f-Cre developed endometrial cancers starting at 1 year. Endometrial cancers exhibited histologic features previously reported in human POLE tumors, and metastasized in all mice (p<0.00001, n=36 vs n=28 controls). Of note, 100% penetrance was achieved through a single (monoallelic) amino acid change, with experimental animals efficiently generated in a single generation. Whole-genome and targeted cancer gene panel sequencing of primary tumors (n=12) and cell lines (n=6) revealed distinctive mutational signatures and the highest nucleotide substitution rates described in murine cancers, with some tumors exceeding 100 base substitutions/Mb. Since most DNA replication errors are repaired by mismatch repair (MMR), and some human tumors harbor POLE mutations and defective MMR (dMMR), we introduced dMMR via conditional Msh2 knockout. This further accelerated tumor progression and mortality (p<0.00001, n=36 Pole alone vs. n=28 Pole+Msh2), revealing clear synergism. In both models, there were robust antitumor immune responses, as evidenced by increased T-cell infiltrates, accelerated tumor growth following T-cell depletion by anti-CD4 antibody, and clinical regression following anti-CTLA4/PDL1 checkpoint therapy. In summary, this is a robust approach to model the high mutational loads intrinsic to human cancer. Expression of PoleP286R in a specific lineage is sufficient to result in cancers with greater ease than previous strategies such as dMMR alone, and with mutational loads in the range of human cancers. The approach may be useful for diverse models and investigations. This abstract is also being presented as Poster A34. Citation Format: Hao-Dong Li, Changzheng Lu, He Zhang, Ileana C. Cuevas, Subhransu S. Sahoo, Yang-Xin Fu, Diego H. Castrillon. Polymerase-mediated ultramutagenesis: A new approach for modeling the high mutational load of human cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR13.