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.

Practical Approaches for the Yeast Saccharomyces cerevisiae Genome Modification.

The yeast S. cerevisiae is a unique genetic object for which a wide range of relatively simple, inexpensive, and non-time-consuming methods have been developed that allow the performing of a wide variety of genome modifications. Among the latter, one can mention point mutations, disruptions and deletions of particular genes and regions of chromosomes, insertion of cassettes for the expression of heterologous genes, targeted chromosomal rearrangements such as translocations and inversions, directed changes in the karyotype (loss or duplication of particular chromosomes, changes in the level of ploidy), mating-type changes, etc. Classical yeast genome manipulations have been advanced with CRISPR/Cas9 technology in recent years that allow for the generation of multiple simultaneous changes in the yeast genome. In this review we discuss practical applications of both the classical yeast genome modification methods as well as CRISPR/Cas9 technology. In addition, we review methods for ploidy changes, including aneuploid generation, methods for mating type switching and directed DSB. Combined with a description of useful selective markers and transformation techniques, this work represents a nearly complete guide to yeast genome modification.

The Increased Amyloidogenicity of Spike RBD and pH-Dependent Binding to ACE2 May Contribute to the Transmissibility and Pathogenic Properties of SARS-CoV-2 Omicron as Suggested by In Silico Study.

SARS-CoV-2 is a rapidly evolving pathogen that has caused a global pandemic characterized by several consecutive waves. Based on epidemiological and NGS data, many different variants of SARS-CoV-2 were described and characterized since the original variant emerged in Wuhan in 2019. Notably, SARS-CoV-2 variants differ in transmissibility and pathogenicity in the human population, although the molecular basis for this difference is still debatable. A significant role is attributed to amino acid changes in the binding surface of the Spike protein to the ACE2 receptor, which may facilitate virus entry into the cell or contribute to immune evasion. We modeled in silico the interaction between Spike RBDs of Wuhan-Hu-1, Delta, and Omicron BA.1 variants and ACE2 at different pHs (pH 5 and pH 7) and showed that the strength of this interaction was higher for the Omicron BA.1 RBD compared to Wuhan-Hu-1 or Delta RBDs and that the effect was more profound at pH 5. This finding is strikingly related to the increased ability of Omicron variants to spread in the population. We also noted that during its spread in the population, SARS-CoV-2 evolved to a more charged, basic composition. We hypothesize that the more basic surface of the Omicron variant may facilitate its spread in the upper respiratory tract but not in the lower respiratory tract, where pH estimates are different. We calculated the amyloidogenic properties of Spike RBDs in different SARS-CoV-2 variants and found eight amyloidogenic regions in the Spike RBDs for each of the variants predicted by the FoldAmyloid program. Although all eight regions were almost identical in the Wuhan to Gamma variants, two of them were significantly longer in both Omicron variants, making the Omicron RBD more amyloidogenic. We discuss how the increased predicted amyloidogenicity of the Omicron variants RBDs may be important for protein stability, influence its interaction with ACE2 and contribute to immune evasion.

Human RAD51 Protein Forms Amyloid-like Aggregates In Vitro.

RAD51 is a central protein of homologous recombination and DNA repair processes that maintains genome stability and ensures the accurate repair of double-stranded breaks (DSBs). In this work, we assessed amyloid properties of RAD51 in vitro and in the bacterial curli-dependent amyloid generator (C-DAG) system. Resistance to ionic detergents, staining with amyloid-specific dyes, polarized microscopy, transmission electron microscopy (TEM), X-ray diffraction and other methods were used to evaluate the properties and structure of RAD51 aggregates. The purified human RAD51 protein formed detergent-resistant aggregates in vitro that had an unbranched cross-ß fibrillar structure, which is typical for amyloids, and were stained with amyloid-specific dyes. Congo-red-stained RAD51 aggregates demonstrated birefringence under polarized light. RAD51 fibrils produced sharp circular X-ray reflections at 4.7 Å and 10 Å, demonstrating that they had a cross-ß structure. Cytoplasmic aggregates of RAD51 were observed in cell cultures overexpressing RAD51. We demonstrated that a key protein that maintains genome stability, RAD51, has amyloid properties in vitro and in the C-DAG system and discussed the possible biological relevance of this observation.

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.

Synthetic Studies toward 1,2,3,3a,4,8b-Hexahydropyrrolo[3,2-b]indole Core. Unusual Fragmentation with 1,2-Aryl Shift.

Base-assisted transformations of 2-(3-oxoindolin-2-yl)acetonitriles were investigated. Unexpectedly, attempted reactions of substrates possessing nonprotected nitrogen atoms were accompanied by unusual extrusions of 2-arylacetonitriles, followed by a 1,2-aryl shift to afford 3-hydroxyindolin-2-ones. On the other hand, the reactions for N-alkyl derivatives of oxoindolines took the expected route by only providing 1,2,3,3a,4,8b-hexahydropyrrolo[3,2-b]indoles.

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.

At the Beginning of the End and in the Middle of the Beginning: Structure and Maintenance of Telomeric DNA Repeats and Interstitial Telomeric Sequences.

Tandem DNA repeats derived from the ancestral (TTAGGG)n run were first detected at chromosome ends of the majority of living organisms, hence the name telomeric DNA repeats. Subsequently, it has become clear that telomeric motifs are also present within chromosomes, and they were suitably called interstitial telomeric sequences (ITSs). It is well known that telomeric DNA repeats play a key role in chromosome stability, preventing end-to-end fusions and precluding the recurrent DNA loss during replication. Recent data suggest that ITSs are also important genomic elements as they confer its karyotype plasticity. In fact, ITSs appeared to be among the most unstable microsatellite sequences as they are highly length polymorphic and can trigger chromosomal fragility and gross chromosomal rearrangements. Importantly, mechanisms responsible for their instability appear to be similar to the mechanisms that maintain the length of genuine telomeres. This review compares the mechanisms of maintenance and dynamic properties of telomeric repeats and ITSs and discusses the implications of these dynamics on genome stability.

Genetic Control of Genomic Alterations Induced in Yeast by Interstitial Telomeric Sequences.

In many organisms, telomeric sequences can be located internally on the chromosome in addition to their usual positions at the ends of the chromosome. In humans, such interstitial telomeric sequences (ITSs) are nonrandomly associated with translocation breakpoints in tumor cells and with chromosome fragile sites (regions of the chromosome that break in response to perturbed DNA replication). We previously showed that ITSs in yeast generated several different types of instability, including terminal inversions (recombination between the ITS and the "true" chromosome telomere) and point mutations in DNA sequences adjacent to the ITS. In the current study, we examine the genetic control of these events. We show that the terminal inversions occur by the single-strand annealing pathway of DNA repair following the formation of a double-stranded DNA break within the ITS. The point mutations induced by the ITS require the error-prone DNA polymerase ζ. Unlike the terminal inversions, these events are not initiated by a double-stranded DNA break, but likely result from the error-prone repair of a single-stranded DNA gap or recruitment of DNA polymerase ζ in the absence of DNA damage.

Quantitative Analysis of the Rates for Repeat-Mediated Genome Instability in a Yeast Experimental System.

Instability of repetitive DNA sequences causes numerous hereditary disorders in humans, the majority of which are associated with trinucleotide repeat expansions. Here, we describe a unique system to study instability of triplet repeats in a yeast experimental setting. Using fluctuation assay and the novel program FluCalc we are able to accurately estimate the rates of large-scale expansions, as well as repeat-mediated mutagenesis and gross chromosomal rearrangements for different repeat sequences.

A Defective mRNA Cleavage and Polyadenylation Complex Facilitates Expansions of Transcribed (GAA)n Repeats Associated with Friedreich's Ataxia.

Expansions of microsatellite repeats are responsible for numerous hereditary diseases in humans, including myotonic dystrophy and Friedreich's ataxia. Whereas the length of an expandable repeat is the main factor determining disease inheritance, recent data point to genomic trans modifiers that can impact the likelihood of expansions and disease progression. Detection of these modifiers may lead to understanding and treating repeat expansion diseases. Here, we describe a method for the rapid, genome-wide identification of trans modifiers for repeat expansion in a yeast experimental system. Using this method, we found that missense mutations in the endoribonuclease subunit (Ysh1) of the mRNA cleavage and polyadenylation complex dramatically increase the rate of (GAA)n repeat expansions but only when they are actively transcribed. These expansions correlate with slower transcription elongation caused by the ysh1 mutation. These results reveal an interplay between RNA processing and repeat-mediated genome instability, confirming the validity of our approach.

Expansion of Interstitial Telomeric Sequences in Yeast.

Telomeric repeats located within chromosomes are called interstitial telomeric sequences (ITSs). They are polymorphic in length and are likely hotspots for initiation of chromosomal rearrangements that have been linked to human disease. Using our S. cerevisiae system to study repeat-mediated genome instability, we have previously shown that yeast telomeric (Ytel) repeats induce various gross chromosomal rearrangements (GCR) when their G-rich strands serve as the lagging strand template for replication (G orientation). Here, we show that interstitial Ytel repeats in the opposite C orientation prefer to expand rather than cause GCR. A tract of eight Ytel repeats expands at a rate of 4 × 10(-4) per replication, ranking them among the most expansion-prone DNA microsatellites. A candidate-based genetic analysis implicates both post-replication repair and homologous recombination pathways in the expansion process. We propose a model for Ytel repeat expansions and discuss its applications for genome instability and alternative telomere lengthening (ALT).

Genome rearrangements caused by interstitial telomeric sequences in yeast.

Interstitial telomeric sequences (ITSs) are present in many eukaryotic genomes and are linked to genome instabilities and disease in humans. The mechanisms responsible for ITS-mediated genome instability are not understood in molecular detail. Here, we use a model Saccharomyces cerevisiae system to characterize genome instability mediated by yeast telomeric (Ytel) repeats embedded within an intron of a reporter gene inside a yeast chromosome. We observed a very high rate of small insertions and deletions within the repeats. We also found frequent gross chromosome rearrangements, including deletions, duplications, inversions, translocations, and formation of acentric minichromosomes. The inversions are a unique class of chromosome rearrangement involving an interaction between the ITS and the true telomere of the chromosome. Because we previously found that Ytel repeats cause strong replication fork stalling, we suggest that formation of double-stranded DNA breaks within the Ytel sequences might be responsible for these gross chromosome rearrangements.

Mismatch repair-independent increase in spontaneous mutagenesis in yeast lacking non-essential subunits of DNA polymerase ε.

Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3' to 5'exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.

The HAL3-PPZ1 dependent regulation of nonsense suppression efficiency in yeast and its influence on manifestation of the yeast prion-like determinant [ISP(+)].

The efficiency of stop codons read-through in yeast is controlled by multiple interactions of genetic and epigenetic factors. In this study, we demonstrate the participation of the Hal3-Ppz1 protein complex in regulation of read-through efficiency and manifestation of non-Mendelian anti-suppressor determinant [ISP(+)]. Over-expression of HAL3 in [ISP(+)] strain causes nonsense suppression, whereas its inactivation displays as anti-suppression of sup35 mutation in [isp(-)] strain. [ISP(+)] strains carrying hal3Delta deletion cannot be cured from [ISP(+)] in the presence of GuHCl. Since Hal3p is a negative regulatory subunit of Ppz1 protein phosphatase, consequences of PPZ1 over-expression and deletion are opposite to those of HAL3. The observed effects are mediated by the catalytic function of Ppz1 and are probably related to the participation of Ppz1 in regulation of eEF1Balpha elongation factor activity. Importantly, [ISP(+)] status of yeast strains is determined by fluctuation in Hal3p level, since [ISP(+)] strains have less Hal3p than their [isp(-)] derivatives obtained by GuHCl treatment. A model considering epigenetic (possibly prion) regulation of Hal3p amount as a mechanism underlying [ISP(+)] status of yeast cell is suggested.

Correlation of somatic hypermutation specificity and A-T base pair substitution errors by DNA polymerase eta during copying of a mouse immunoglobulin kappa light chain transgene.

To test the hypothesis that inaccurate DNA synthesis by mammalian DNA polymerase eta (pol eta) contributes to somatic hypermutation (SHM) of Ig genes, we measured the error specificity of mouse pol eta during synthesis of each strand of a mouse Ig kappa light chain transgene. We then compared the results to the base substitution specificity of SHM of this same gene in the mouse. The in vitro and in vivo base substitution spectra shared a number of common features. A highly significant correlation was observed for overall substitutions at A-T pairs but not for substitutions at G-C pairs. Sixteen mutational hotspots at A-T pairs observed in vivo were also found in spectra generated by mouse pol eta in vitro. The correlation was strongest for errors made by pol eta during synthesis of the non-transcribed strand, but it was also observed for synthesis of the transcribed strand. These facts, and the distribution of substitutions generated in vivo, support the hypothesis that pol eta contributes to SHM of Ig genes at A-T pairs via short patches of low fidelity DNA synthesis of both strands, but with a preference for the non-transcribed strand.

Novel non-Mendelian determinant involved in the control of translation accuracy in Saccharomyces cerevisiae.

Two cytoplasmically inherited determinants related by their manifestation to the control of translation accuracy were previously described in yeast. Cells carrying one of them, [PSI(+)], display a nonsense suppressor phenotype and contain a prion form of the Sup35 protein. Another element, [PIN(+)], determines the probability of de novo generation of [PSI(+)] and results from a prion form of several proteins, which can be functionally unrelated to Sup35p. Here we describe a novel nonchromosomal determinant related to the SUP35 gene. This determinant, designated [ISP(+)], was identified as an antisuppressor of certain sup35 mutations. We observed its loss upon growth on guanidine hydrochloride and subsequent spontaneous reappearance with high frequency. The reversible curability of [ISP(+)] resembles the behavior of yeast prions. However, in contrast to known prions, [ISP(+)] does not depend on the chaperone protein Hsp104. Though manifestation of both [ISP(+)] and [PSI(+)] is related to the SUP35 gene, the maintenance of [ISP(+)] does not depend on the prionogenic N-terminal domain of Sup35p and Sup35p is not aggregated in [ISP(+)] cells, thus ruling out the possibility that [ISP(+)] is a specific form of [PSI(+)]. We hypothesize that [ISP(+)] is a novel prion involved in the control of translation accuracy in yeast.