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Abstract Staphylococcus aureus is a common cause of diverse infections, ranging from superficial to invasive, affecting both humans and animals. The widespread use of antibiotics in clinical treatments has led to the emergence of antibiotic‐resistant strains and small colony variants. This surge presents a significant challenge in eliminating infections and undermines the efficacy of available treatments. The bacterial Save Our Souls (SOS) response, triggered by genotoxic stressors, encompasses host immune defenses and antibiotics, playing a crucial role in bacterial survival, invasiveness, virulence, and drug resistance. Accumulating evidence underscores the pivotal role of the SOS response system in the pathogenicity of S. aureus. Inhibiting this system offers a promising approach for effective bactericidal treatments and curbing the evolution of antimicrobial resistance. Here, we provide a comprehensive review of the activation, impact, and key proteins associated with the SOS response in S. aureus. Additionally, perspectives on therapeutic strategies targeting the SOS response for S. aureus, both individually and in combination with traditional antibiotics are proposed.

The Lyme disease spirochete, Borrelia burgdorferi, uses antigenic variation as a strategy to evade the host’s acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE, and the importance of sequence homology in determining the location of recombination, despite RecA’s dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5′ end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions.

Highlights • An alternative ultraviolet (UV) excision repair pathway operates in B. subtilis sporangia. • The UV-edonuclease YwjD is a versatile repair protein that incise DNA damaged with a wide range of pyrimidine photodimers. • Exonuclease YpcP and low fidelity YqjH and YqjW DNA polymerases complement the incision repair event started by YwjD. • YwjD, YpcP and YqjH/YqjW, conform a novel error-prone repair pathway that protect B. subtilis sporangia from UV light. Abstract It has been shown that mutation frequency decline (Mfd) and nucleotide excision repair (NER) deficiencies promote UV-induced mutagenesis in B. subtilis sporangia. As replication is halted in sporulating B. subtilis cells, in this report, we investigated if this response may result from an error-prone repair event involving the UV-endonuclease YwjD and low fidelity (LF) DNA synthesis. Accordingly, disruption of YwjD generated B. subtilis sporangia that were more susceptible to UV-C radiation than sporangia of the WT strain and such susceptibility increased even more after the single or simultaneous inactivation of the LF DNA polymerases YqjH and YqjW. To further explore this concept, functional His 6 -tagged YwjD and Y-DNA polymerases YqjH and YqjW were produced and purified to homogeneity. In vitro repair assays showed that YwjD hydrolyzed the phosphodiester bond immediately located 5´-end of a ds -DNA substrate bearing either, cyclobutane pyrimidine dimers (CPD), 6-4 photoproducts (6-4 PD) or Dewar isomers (DWI). Notably, the 6-4 PD and DWI but not the CPDs repair intermediaries of YwjD were efficiently processed by the LF polymerase YqjH suggesting that an additional 5′→3′ exonuclease event was necessary to process PD. Accordingly, the LF polymerase YqjW efficiently processed the incision-excision repair products derived from YwjD and exonuclease YpcP attack over CPD-containing DNA. In summary, our results unveiled a novel non-canonical repair pathway that employs YwjD to incise PD-containing DNA and low fidelity synthesis contributing thus to mutagenesis, survival and spore morphogenesis in B. subtilis. [ABSTRACT FROM AUTHOR]

Summary The Lyme disease spirochete, Borrelia burgdorferi , uses antigenic variation as a strategy to evade the host’s acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE , and the importance of sequence homology in determining the location of recombination, despite RecA’s dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5′ end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions. [ABSTRACT FROM AUTHOR]

Upon DNA damage or other stressors, the tumor suppressor p53 is activated, leading to transient expression of the cyclin-dependent kinase inhibitor (CKI) p21. This either triggers momentary G1 cell cycle arrest or leads to a chronic state of senescence or apoptosis, a form of genome guardianship. In the clinic, the presence of p21 has been considered an indicator of wildtype p53 activity. However, recent evidence suggests that p21 also acts as an oncogenic factor in a p53-deficient environment. Here, we discuss the controversial aspects of the two-faced involvement of p21 in cancer and speculate on how this new information may increase our understanding of its role in cancer pathogenesis. Prevailing notions indicate that p21 might also act as antiapoptotic agent, which may have relevant implications for future therapeutic strategies. [ABSTRACT FROM AUTHOR]

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential. [ABSTRACT FROM AUTHOR]

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism carried out by low-fidelity DNA polymerases that bypass DNA lesions, which overcomes replication stalling. Despite the miscoding nature of most common DNA lesions, several of them are bypassed in mammalian cells in a relatively accurate manner, which plays a key role maintaining a low mutation load. Whereas it is generally agreed that TLS across the major UV and sunlight induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), is accurate, there were conflicting reports on whether the same is true for the thymine–thymine pyrimidine–pyrimidone(6-4) ultraviolet light photoproduct (TT6-4PP), which represents the second most common class of UV lesions. Using a TLS assay system based on gapped plasmids carrying site-specific TT6-4PP lesions in defined sequence contexts we show that the DNA sequence context markedly affected both the extent and accuracy of TLS. The sequence exhibiting higher TLS exhibited also higher error-frequency, caused primarily by semi-targeted mutations, at the nearest nucleotides flanking the lesion. Our results resolve the discrepancy reported on TLS across TT6-4PP, and suggest that TLS is more accurate in human cells than in mouse cells. [ABSTRACT FROM AUTHOR]

Abstract: Disease progression in myeloid malignancies results from the accumulation of “mutations” in genes that control cellular growth and differentiation. Many types of genetic alterations have been identified in myeloid diseases. However, the mechanism(s) by which these cells acquire genetic alterations or “Genomic instability”, is less well understood. Increasing evidence suggests that the genetic changes in myeloid malignancies lead to increased production of endogenous sources of DNA damage, such as, reactive oxygen species (ROS). The fusion gene BCR–ABL in chronic myeloid leukemia (CML), FLT3/ITD in acute myeloid leukemia (AML), and RAS mutations in myelodysplastic syndromes (MDS)/myeloproliferative diseases (MPD) result in ROS production. Increased ROS can drive a cycle of genomic instability leading to DNA double strand breaks (DSBs) and altered repair that can lead to acquisition of genomic changes. Evidence is coming to light that defects in a main repair pathway for DSBs, non-homologous end-joining (NHEJ), lead to up-regulation of alternative or “back-up” repair that can create chromosomal deletions and translocations. This article will review evidence for activation of RAS/PI3K/STAT pathways, that lead to increased ROS, DNA damage and defective repair in myeloid diseases, a mechanism for acquisition of additional mutations that can drive disease progression. [Copyright &y& Elsevier]

Abstract: Xeroderma pigmentosum variant (XPV) patients carry germ-line mutations in DNA polymerase η (polη), a major translesion DNA synthesis (TLS) polymerase, and exhibit severe sunlight sensitivity and high predisposition to skin cancer. Using a quantitative TLS assay system based on gapped plasmids we analyzed TLS across a site-specific TT CPD (thymine–thymine cyclobutane pyrimidine dimer) or TT 6-4 PP (thymine–thymine 6-4 photoproduct) in three pairs of polη-proficient and deficient human cells. TLS across the TT CPD lesion was reduced by 2.6–4.4-fold in cells lacking polη, and exhibited a strong 6–17-fold increase in mutation frequency at the TT CPD. All targeted mutations (74%) in polη-deficient cells were opposite the 3′T of the CPD, however, a significant fraction (23%) were semi-targeted to the nearest nucleotides flanking the CPD. Deletions and insertions were observed at a low frequency, which increased in the absence of polη, consistent with the formation of double strand breaks due to defective TLS. TLS across TT 6-4 PP was about twofold lower than across CPD, and was marginally reduced in polη-deficient cells. TLS across TT 6-4 PP was highly mutagenic (27–63%), with multiple mutations types, and no significant difference between cells with or without polη. Approximately 50% of the mutations formed were semi-targeted, of which 84–93% were due to the insertion of an A opposite the template G 5′ to the 6-4 PP. These results, which are consistent with the UV hyper-mutability of XPV cells, highlight the critical role of polη in error-free TLS across CPD in human cells, and suggest a potential involvement, although minor, of polη in TLS across 6-4 PP under some conditions. [Copyright &y& Elsevier]

Purpose: To study nonhomologous end joining in extracts of two lymphoblastoid cell lines derived from patients with late radionecrosis after radiotherapy. Both cell lines were previously shown to exhibit impaired rejoining of DNA double-strand breaks in a pulse-field gel electrophoresis assay. Methods and Materials: We used a cell-free system and quantitative real-time polymerase chain reaction, as well as sequencing analysis of end joining products. Results: Paradoxically, extracts of the two cell lines display increased rates of in vitro end joining of noncohesive termini compared with normal cell extracts. This increase was seen in the absence of added deoxyribonucleoside triphosphates and was sensitive to inhibition by wortmannin. Sequencing of the joined products revealed that, despite increased rates of end joining, the process was error prone with a greater frequency of deletions compared with that observed in normal controls. Conclusion: These findings are consistent with the suggestion that a promiscuous, deletion-prone abnormality of nonhomologous end joining might underpin the predisposition of certain radiotherapy patients to late radionecrosis. We hypothesize that some individuals might harbor subclinical defects in nonhomologous end joining that clinically manifest on challenge with high-dose radiation. Because both quantitative and qualitative aspects of end joining have demonstrably been influenced, we recommend that the study of patient samples should involve a combination of quantitative methods (e.g., quantitative real-time polymerase chain reaction), sequencing analysis, and a comparison of multiple join types. [Copyright &y& Elsevier]

Affinity maturation of the humoral response is mediated by somatic hypermutation of the immunoglobulin (Ig) genes and selection of higher-affinity B cell clones. Activation-induced cytidine deaminase (AID) is the first of a complex series of proteins that introduce these point mutations into variable regions of the Ig genes. AID deaminates deoxycytidine residues in single-stranded DNA to deoxyuridines, which are then processed by DNA replication, base excision repair (BER), or mismatch repair (MMR). In germinal center B cells, MMR, BER, and other factors are diverted from their normal roles in preserving genomic integrity to increase diversity within the Ig locus. Both AID and these components of an emerging error-prone mutasome are regulated on many levels by complex mechanisms that are only beginning to be elucidated. [ABSTRACT FROM AUTHOR]

The PSO3 gene of Saccharomyces cerevisiae was molecularly cloned by complementing the cold-sensitivity phenotype of a pso3-1 mutant and was found to be allelic to RNR4, encoding one of the two DNA damage-inducible small subunits of the ribonucleotide reductase (RNR) complex. Compared to a rnr4Δ mutant that allows only very little mutation induction at very low doses of 254nm ultraviolet light (UVC), the pso3-1 mutant allele confers leakiness in that it permits some DNA damage-induced mutagenesis at low doses of UVC. Similarly, the pso3 mutant is slightly less sensitive to UVC than an rnr4Δ mutant. Cloning and sequencing of the RNR4 locus of the pso3-1 mutant revealed that its intermediate phenotype is attributable to a G → A transition at nucleotide 352, leading to replacement of glycine by arginine [G118R] in the mutant’s protein. Both RNR4 mutant alleles confer significantly less sensitivity to UVC than mutant alleles of non-UVC-mutable REV3, indicating that, apart from nucleotide excision repair, RAD6-dependent error-free DNA repair may still be functional. The phenotype of a strongly reduced UVC-induced mutagenesis for rnr4 mutant alleles has not yet been described; it suggests the importance of this gene for a fully functional RNR providing correct amounts of DNA precursor molecules, thereby, allowing translesion synthesis (error-prone) of UVC-damaged DNA. Stationary phase cells of the rnr4Δ mutant, but not of the original pso3-1 mutant, are swollen with a fourfold to eightfold increase in volume. The central role of RNR in DNA precursor metabolism and its complex regulation allow for several modes of suppression that may influence the phenotypes of RNR4 mutants, especially those containing the leaky pso3-1 mutant allele. [ABSTRACT FROM AUTHOR]

Abstract: DNA lesions that have escaped DNA repair are tolerated via translesion DNA synthesis (TLS), carried out by specialized error-prone DNA polymerases. To evaluate the robustness of the TLS system in human cells, we examined its ability to cope with foreign non-DNA stretches of 3 or 12 methylene residues, using a gap-lesion plasmid assay system. We found that both the trimethylene and dodecamethylene inserts were bypassed with significant efficiencies in human cells, using both misinsertion and misalignment mechanisms. TLS across these non-DNA segments was aphidicolin-sensitive, and did not require polη. In vitro primer extension assays showed that purified polη, polκ and polι were each capable of inserting each of the four nucleotides opposite the trimethylene chain, but only polη and polκ could fully bypass it. Polη and polι, but not polκ, could also insert each of the four nucleotides opposite the dodecamethylene chain, but all three polymerases were severely blocked by this lesion. The ability of TLS polymerases to insert nucleotides opposite a hydrocarbon chain, despite the lack of any similarity to DNA, suggests that they may act via a mode of transient and local template-independent polymerase activity, and highlights the robustness of the TLS system in human cells. [Copyright &y& Elsevier]

Nuclear protein coding sequences from gymnosperms are currently scarce. We have determined 4 kb of nuclear protein coding sequences from gymnosperms and have collected and analyzed >60 kb of nuclear sequences from gymnosperms and nonspermatophytes in order to better understand processes influencing genome evolution in plants. We show that conifers possess both biased and nonbiased genes with respect to GC content, as found in monocots, suggesting that the common ancestor of conifers and monocots may have possessed both biased and nonbiased genes. The lack of biased genes in dicots is suggested to be a derived character for this lineage. We present a simple but speculative model of land-plant genome evolution which considers changes in GC bias and CpG frequency, respectively, as independent processes and which can account for several puzzling aspects of observed nucleotide frequencies in plant genes. [ABSTRACT FROM AUTHOR]

The original pso3-1 mutant isolate of the yeast Saccharomyces cerevisiae exhibits a pleiotropic mutagen-sensitivity phenotype that includes sensitivity to UVA-activated 3-carbethoxypsoralen, to UVC-light, to mono- and bi-functional nitrogen mustard, to paraquat, and to cadmium; on the other hand, it shows hyper-resistance (HYR) to nitrosoguanidine when compared to established wild-type strains. Also, the original pso3-1 mutant exhibits a low UVC-induced mutability and mitotic gene conversion and a high rate of spontaneous and UVC-induced petite mutations. Since the HYR to the nitrosoguanidine (MNNG) phenotype resembles that of low glutathione-containing yeast cells, the original pso3-1 mutant was crossed to a gsh1 knock-out mutant that lacks the enzyme for the first step in glutathione biosynthesis and the resulting diploid was tested for complementation. While there was none for HYR to nitrosoguanidine, and other low glutathione-related phenotypes, some other phenotypic characteristics of pso3-1, e.g. UVC sensitivity and UVC-induced mutability were restored to a wild-type level. Tetrad analysis of a diploid derived from a cross of the original haploid pso3-1 isolate with a repair-proficient, normal glutathione-containing, PSO3 GSH1 wild-type led to the separation of a leaky gsh1 mutation phenotype from that of the repair-deficient pso3-1 phenotype. Linkage studies by tetrad and random spore analyses indicated no linkage of the two genes. This shows that the low glutathione content in the original pso3-1 isolate is due to a second, additional, mutation in the GSH1 locus and is unrelated to the pso3-1 mutation. Thus, the original pso3-1 isolate is a pso3-1 gsh1 double mutant with most of the particular characteristics of the pleiotropic sensitivity phenotype contributed by either the pso3-1 or the gsh1-leaky mutant allele. The expression of a few phenotypic characteristics of pso3, however, were most pronounced in pso3-1 mutants with a low glutathione pool. [ABSTRACT FROM AUTHOR]