G-Quadruplex Forming DNA Sequence Context Is Enriched around Points of Somatic Mutations in a Subset of Multiple Myeloma Patients.

Multiple myeloma (MM) is the second most common hematological malignancy, which remains incurable despite recent advances in treatment strategies. Like other forms of cancer, MM is characterized by genomic instability, caused by defects in DNA repair. Along with mutations in DNA repair genes and genotoxic drugs used to treat MM, non-canonical secondary DNA structures (four-stranded G-quadruplex structures) can affect accumulation of somatic mutations and chromosomal abnormalities in the tumor cells of MM patients. Here, we tested the hypothesis that G-quadruplex structures may influence the distribution of somatic mutations in the tumor cells of MM patients. We sequenced exomes of normal and tumor cells of 11 MM patients and analyzed the data for the presence of G4 context around points of somatic mutations. To identify molecular mechanisms that could affect mutational profile of tumors, we also analyzed mutational signatures in tumor cells as well as germline mutations for the presence of specific SNPs in DNA repair genes or in genes regulating G-quadruplex unwinding. In several patients, we found that sites of somatic mutations are frequently located in regions with G4 context. This pattern correlated with specific germline variants found in these patients. We discuss the possible implications of these variants for mutation accumulation and specificity in MM and propose that the extent of G4 context enrichment around somatic mutation sites may be a novel metric characterizing mutational processes in tumors.

Properties and Mechanisms of Deletions, Insertions, and Substitutions in the Evolutionary History of SARS-CoV-2.

SARS-CoV-2 has accumulated many mutations since its emergence in late 2019. Nucleotide substitutions leading to amino acid replacements constitute the primary material for natural selection. Insertions, deletions, and substitutions appear to be critical for coronavirus's macro- and microevolution. Understanding the molecular mechanisms of mutations in the mutational hotspots (positions, loci with recurrent mutations, and nucleotide context) is important for disentangling roles of mutagenesis and selection. In the SARS-CoV-2 genome, deletions and insertions are frequently associated with repetitive sequences, whereas C>U substitutions are often surrounded by nucleotides resembling the APOBEC mutable motifs. We describe various approaches to mutation spectra analyses, including the context features of RNAs that are likely to be involved in the generation of recurrent mutations. We also discuss the interplay between mutations and natural selection as a complex evolutionary trend. The substantial variability and complexity of pipelines for the reconstruction of mutations and the huge number of genomic sequences are major problems for the analyses of mutations in the SARS-CoV-2 genome. As a solution, we advocate for the development of a centralized database of predicted mutations, which needs to be updated on a regular basis.

CRISPR/Cas9 as a Mutagenic Factor.

The discovery of the CRISPR/Cas9 microbial adaptive immune system has revolutionized the field of genetics, by greatly enhancing the capacity for genome editing. CRISPR/Cas9-based editing starts with DNA breaks (or other lesions) predominantly at target sites and, unfortunately, at off-target genome sites. DNA repair systems differing in accuracy participate in establishing desired genetic changes but also introduce unwanted mutations, that may lead to hereditary, oncological, and other diseases. New approaches to alleviate the risks associated with genome editing include attenuating the off-target activity of editing complex through the use of modified forms of Cas9 nuclease and single guide RNA (sgRNA), improving delivery methods for sgRNA/Cas9 complex, and directing DNA lesions caused by the sgRNA/Cas9 to non-mutagenic repair pathways. Here, we have described CRISPR/Cas9 as a new powerful mutagenic factor, discussed its mutagenic properties, and reviewed factors influencing the mutagenic activity of CRISPR/Cas9.

Detection of Primary DNA Lesions by Transient Changes in Mating Behavior in Yeast Saccharomyces cerevisiae Using the Alpha-Test.

Spontaneous or induced DNA lesions can result in stable gene mutations and chromosomal aberrations due to their inaccurate repair, ultimately resulting in phenotype changes. Some DNA lesions per se may interfere with transcription, leading to temporary phenocopies of mutations. The direct impact of primary DNA lesions on phenotype before their removal by repair is not well understood. To address this question, we used the alpha-test, which allows for detecting various genetic events leading to temporary or hereditary changes in mating type α→a in heterothallic strains of yeast Saccharomyces cerevisiae. Here, we compared yeast strains carrying mutations in DNA repair genes, mismatch repair (pms1), base excision repair (ogg1), and homologous recombination repair (rad52), as well as mutagens causing specific DNA lesions (UV light and camptothecin). We found that double-strand breaks and UV-induced lesions have a stronger effect on the phenotype than mismatches and 8-oxoguanine. Moreover, the loss of the entire chromosome III leads to an immediate mating type switch α→a and does not prevent hybridization. We also evaluated the ability of primary DNA lesions to persist through the cell cycle by assessing the frequency of UV-induced inherited and non-inherited genetic changes in asynchronous cultures of a wild-type (wt) strain and in a cdc28-4 mutant arrested in the G1 phase. Our findings suggest that the phenotypic manifestation of primary DNA lesions depends on their type and the stage of the cell cycle in which it occurred.

Polymorphism of Saccharomyces cerevisiae Strains in DNA Metabolism Genes.

Baker's yeast, S. cerevisiae, is an excellent model organism exploited for molecular genetic studies of the mechanisms of genome stability in eukaryotes. Genetic peculiarities of commonly used yeast strains impact the processes of DNA replication, repair, and recombination (RRR). We compared the genomic DNA sequence variation of the five strains that are intensively used for RRR studies. We used yeast next-generation sequencing data to detect the extent and significance of variation in 183 RRR genes. We present a detailed analysis of the differences that were found even in closely related strains. Polymorphisms of common yeast strains should be considered when interpreting the outcomes of genome stability studies, especially in cases of discrepancies between laboratories describing the same phenomena.

DNA Polymerase ζ without the C-Terminus of Catalytic Subunit Rev3 Retains Characteristic Activity, but Alters Mutation Specificity of Ultraviolet Radiation in Yeast.

DNA polymerase ζ (pol ζ) plays a central role in replicating damaged genomic DNA. When DNA synthesis stalls at a lesion, it participates in translesion DNA synthesis (TLS), which helps replication proceed. TLS prevents cell death at the expense of new mutations. The current model indicates that pol ζ-dependent TLS events are mediated by Pol31/Pol32 pol ζ subunits, which are shared with replicative polymerase pol δ. Surprisingly, we found that the mutant rev3-ΔC in yeast, which lacks the C-terminal domain (CTD) of the catalytic subunit of pol ζ and, thus, the platform for interaction with Pol31/Pol32, retains most pol ζ functions. To understand the underlying mechanisms, we studied TLS in normal templates or templates with abasic sites in vitro in primer extension reactions with purified four-subunit pol ζ versus pol ζ with Rev3-ΔC. We also examined the specificity of ultraviolet radiation (UVR)-induced mutagenesis in the rev3-ΔC strains. We found that the absence of Rev3 CTD reduces activity levels, but does not alter the basic biochemical properties of pol ζ, and alters the mutation spectrum only at high doses of UVR, alluding to the existence of mechanisms of recruitment of pol ζ to UVR-damaged sites independent of the interaction of Pol31/Pol32 with the CTD of Rev3.

Partners in crime: Tbf1 and Vid22 promote expansions of long human telomeric repeats at an interstitial chromosome position in yeast.

In humans, telomeric repeats (TTAGGG)n are known to be present at internal chromosomal sites. These interstitial telomeric sequences (ITSs) are an important source of genomic instability, including repeat length polymorphism, but the molecular mechanisms responsible for this instability remain to be understood. Here, we studied the mechanisms responsible for expansions of human telomeric (Htel) repeats that were artificially inserted inside a yeast chromosome. We found that Htel repeats in an interstitial chromosome position are prone to expansions. The propensity of Htel repeats to expand depends on the presence of a complex of two yeast proteins: Tbf1 and Vid22. These two proteins are physically bound to an interstitial Htel repeat, and together they slow replication fork progression through it. We propose that slow progression of the replication fork through the protein complex formed by the Tbf1 and Vid22 partners at the Htel repeat cause DNA strand slippage, ultimately resulting in repeat expansions.

Genome Instability in Multiple Myeloma: Facts and Factors.

Multiple myeloma (MM) is a malignant neoplasm of terminally differentiated immunoglobulin-producing B lymphocytes called plasma cells. MM is the second most common hematologic malignancy, and it poses a heavy economic and social burden because it remains incurable and confers a profound disability to patients. Despite current progress in MM treatment, the disease invariably recurs, even after the transplantation of autologous hematopoietic stem cells (ASCT). Biological processes leading to a pathological myeloma clone and the mechanisms of further evolution of the disease are far from complete understanding. Genetically, MM is a complex disease that demonstrates a high level of heterogeneity. Myeloma genomes carry numerous genetic changes, including structural genome variations and chromosomal gains and losses, and these changes occur in combinations with point mutations affecting various cellular pathways, including genome maintenance. MM genome instability in its extreme is manifested in mutation kataegis and complex genomic rearrangements: chromothripsis, templated insertions, and chromoplexy. Chemotherapeutic agents used to treat MM add another level of complexity because many of them exacerbate genome instability. Genome abnormalities are driver events and deciphering their mechanisms will help understand the causes of MM and play a pivotal role in developing new therapies.

The Study of Cancer Susceptibility Genes.

"…most complex, new direction for cancer medicine is to integrate our understanding of aberrant genes and pathways to explain the behavior of cancer as a whole, thereby renewing the cycle of knowledge, discovery and therapeutic intervention [...].

DNA Methylation, Deamination, and Translesion Synthesis Combine to Generate Footprint Mutations in Cancer Driver Genes in B-Cell Derived Lymphomas and Other Cancers.

Cancer genomes harbor numerous genomic alterations and many cancers accumulate thousands of nucleotide sequence variations. A prominent fraction of these mutations arises as a consequence of the off-target activity of DNA/RNA editing cytosine deaminases followed by the replication/repair of edited sites by DNA polymerases (pol), as deduced from the analysis of the DNA sequence context of mutations in different tumor tissues. We have used the weight matrix (sequence profile) approach to analyze mutagenesis due to Activation Induced Deaminase (AID) and two error-prone DNA polymerases. Control experiments using shuffled weight matrices and somatic mutations in immunoglobulin genes confirmed the power of this method. Analysis of somatic mutations in various cancers suggested that AID and DNA polymerases η and θ contribute to mutagenesis in contexts that almost universally correlate with the context of mutations in A:T and G:C sites during the affinity maturation of immunoglobulin genes. Previously, we demonstrated that AID contributes to mutagenesis in (de)methylated genomic DNA in various cancers. Our current analysis of methylation data from malignant lymphomas suggests that driver genes are subject to different (de)methylation processes than non-driver genes and, in addition to AID, the activity of pols η and θ contributes to the establishment of methylation-dependent mutation profiles. This may reflect the functional importance of interplay between mutagenesis in cancer and (de)methylation processes in different groups of genes. The resulting changes in CpG methylation levels and chromatin modifications are likely to cause changes in the expression levels of driver genes that may affect cancer initiation and/or progression.

Compensation for the absence of the catalytically active half of DNA polymerase ε in yeast by positively selected mutations in CDC28.

Current eukaryotic replication models postulate that leading and lagging DNA strands are replicated predominantly by dedicated DNA polymerases. The catalytic subunit of the leading strand DNA polymerase ε, Pol2, consists of two halves made of two different ancestral B-family DNA polymerases. Counterintuitively, the catalytically active N-terminal half is dispensable, while the inactive C-terminal part is required for viability. Despite extensive studies of yeast Saccharomyces cerevisiae strains lacking the active N-terminal half, it is still unclear how these strains survive and recover. We designed a robust method for constructing mutants with only the C-terminal part of Pol2. Strains without the active polymerase part show severe growth defects, sensitivity to replication inhibitors, chromosomal instability, and elevated spontaneous mutagenesis. Intriguingly, the slow-growing mutant strains rapidly accumulate fast-growing clones. Analysis of genomic DNA sequences of these clones revealed that the adaptation to the loss of the catalytic N-terminal part of Pol2 occurs by a positive selection of mutants with improved growth. Elevated mutation rates help generate sufficient numbers of these variants. Single nucleotide changes in the cell cycle-dependent kinase gene, CDC28, improve the growth of strains lacking the N-terminal part of Pol2, and rescue their sensitivity to replication inhibitors and, in parallel, lower mutation rates. Our study predicts that changes in mammalian homologs of cyclin-dependent kinases may contribute to cellular responses to the leading strand polymerase defects.

Correction: Pavlov, Y.I., et al. DNA Polymerases at the Eukaryotic Replication Fork Thirty Years After: Connection to Cancer. Cancers 2020, 12, 3489.

The authors wish to make the following corrections to this paper [...].

DNA Polymerases at the Eukaryotic Replication Fork Thirty Years after: Connection to Cancer.

Recent studies on tumor genomes revealed that mutations in genes of replicative DNA polymerases cause a predisposition for cancer by increasing genome instability. The past 10 years have uncovered exciting details about the structure and function of replicative DNA polymerases and the replication fork organization. The principal idea of participation of different polymerases in specific transactions at the fork proposed by Morrison and coauthors 30 years ago and later named "division of labor," remains standing, with an amendment of the broader role of polymerase δ in the replication of both the lagging and leading DNA strands. However, cancer-associated mutations predominantly affect the catalytic subunit of polymerase ε that participates in leading strand DNA synthesis. We analyze how new findings in the DNA replication field help elucidate the polymerase variants' effects on cancer.

Comment on "A commensal strain of Staphylococcus epidermidis protects against skin neoplasia" by Nakatsuji et al.

A recent article in Science Advances described the striking discovery that the commensal Staphylococcus epidermidis strain MO34 displays antimicrobial and antitumor activities by producing a small molecule, identified as the nucleobase analog 6-N-hydroxylaminopurine (6-HAP). However, in contradiction to the literature, the authors claimed that 6-HAP is nonmutagenic and proposed that the toxic effect of 6-HAP results from its ability to inhibit, in its base form, DNA synthesis. To resolve the discrepancy, we proved by genetic experiments with bacteria and yeast that extracts of MO34 do contain a mutagenic compound whose effects are identical to chemically synthesized 6-HAP. The MO34 extract induced the same mutation spectrum as authentic 6-HAP. Notably, the toxic and mutagenic effects of both synthetic and MO34-derived 6-HAP depended on conversion to the corresponding nucleotide. The nucleobase 6-HAP does not inhibit DNA synthesis in vitro, and we conclude that 6-HAP exerts its biological activity when incorporated into DNA.

Nucleotide Weight Matrices Reveal Ubiquitous Mutational Footprints of AID/APOBEC Deaminases in Human Cancer Genomes.

Cancer genomes accumulate nucleotide sequence variations that number in the tens of thousands per genome. A prominent fraction of these mutations is thought to arise as a consequence of the off-target activity of DNA/RNA editing cytosine deaminases. These enzymes, collectively called activation induced deaminase (AID)/APOBECs, deaminate cytosines located within defined DNA sequence contexts. The resulting changes of the original C:G pair in these contexts (mutational signatures) provide indirect evidence for the participation of specific cytosine deaminases in a given cancer type. The conventional method used for the analysis of mutable motifs is the consensus approach. Here, for the first time, we have adopted the frequently used weight matrix (sequence profile) approach for the analysis of mutagenesis and provide evidence for this method being a more precise descriptor of mutations than the sequence consensus approach. We confirm that while mutational footprints of APOBEC1, APOBEC3A, APOBEC3B, and APOBEC3G are prominent in many cancers, mutable motifs characteristic of the action of the humoral immune response somatic hypermutation enzyme, AID, are the most widespread feature of somatic mutation spectra attributable to deaminases in cancer genomes. Overall, the weight matrix approach reveals that somatic mutations are significantly associated with at least one AID/APOBEC mutable motif in all studied cancers.

Iron-Sulfur Clusters in DNA Polymerases and Primases of Eukaryotes.

Research during the past decade witnessed the discovery of [4Fe-4S] clusters in several members of the eukaryotic DNA replication machinery. The presence of clusters was confirmed by UV-visible absorption, electron paramagnetic resonance spectroscopy, and metal analysis for primase and the B-family DNA polymerases δ and ζ. The crystal structure of primase revealed that the [4Fe-4S] cluster is buried inside the protein and fulfills a structural role. Although [4Fe-4S] clusters are firmly established in the C-terminal domains of catalytic subunits of DNA polymerases δ and ζ, no structures are currently available and their precise roles have not been ascertained. The [4Fe-4S] clusters in the polymerases and primase play a structural role ensuring proper protein folding and stability. In DNA polymerases δ and ζ, they can potentially play regulatory role by sensing hurdles during DNA replication and assisting with DNA polymerase switches by oscillation between oxidized-reduced states.

Activity and fidelity of human DNA polymerase α depend on primer structure.

DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA primers with higher efficiency but lower processivity than it did with RNA and chimeric primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.

DNA polymerase η mutational signatures are found in a variety of different types of cancer.

DNA polymerase (pol) η is a specialized error-prone polymerase with at least two quite different and contrasting cellular roles: to mitigate the genetic consequences of solar UV irradiation, and promote somatic hypermutation in the variable regions of immunoglobulin genes. Misregulation and mistargeting of pol η can compromise genome integrity. We explored whether the mutational signature of pol η could be found in datasets of human somatic mutations derived from normal and cancer cells. A substantial excess of single and tandem somatic mutations within known pol η mutable motifs was noted in skin cancer as well as in many other types of human cancer, suggesting that somatic mutations in A:T bases generated by DNA polymerase η are a common feature of tumorigenesis. Another peculiarity of pol ηmutational signatures, mutations in YCG motifs, led us to speculate that error-prone DNA synthesis opposite methylated CpG dinucleotides by misregulated pol η in tumors might constitute an additional mechanism of cytosine demethylation in this hypermutable dinucleotide.

Mutational signatures and mutable motifs in cancer genomes.

Cancer is a genetic disorder, meaning that a plethora of different mutations, whether somatic or germ line, underlie the etiology of the 'Emperor of Maladies'. Point mutations, chromosomal rearrangements and copy number changes, whether they have occurred spontaneously in predisposed individuals or have been induced by intrinsic or extrinsic (environmental) mutagens, lead to the activation of oncogenes and inactivation of tumor suppressor genes, thereby promoting malignancy. This scenario has now been recognized and experimentally confirmed in a wide range of different contexts. Over the past decade, a surge in available sequencing technologies has allowed the sequencing of whole genomes from liquid malignancies and solid tumors belonging to different types and stages of cancer, giving birth to the new field of cancer genomics. One of the most striking discoveries has been that cancer genomes are highly enriched with mutations of specific kinds. It has been suggested that these mutations can be classified into 'families' based on their mutational signatures. A mutational signature may be regarded as a type of base substitution (e.g. C:G to T:A) within a particular context of neighboring nucleotide sequence (the bases upstream and/or downstream of the mutation). These mutational signatures, supplemented by mutable motifs (a wider mutational context), promise to help us to understand the nature of the mutational processes that operate during tumor evolution because they represent the footprints of interactions between DNA, mutagens and the enzymes of the repair/replication/modification pathways.