Protein innovation through template switching in the Saccharomyces cerevisiae lineage

DNA polymerase template switching between short, non-identical inverted repeats (IRs) is a genetic mechanism that leads to the homogenization of IR arms and to IR spacer inversion, which cause multinucleotide mutations (MNMs). It is unknown if and how template switching affects gene evolution. In this study, we performed a phylogenetic analysis to determine the effect of template switching between IR arms on coding DNA of Saccharomyces cerevisiae. To achieve this, perfect IRs that co-occurred with MNMs between a strain and its parental node were identified in S. cerevisiae strains. We determined that template switching introduced MNMs into 39 protein-coding genes through S. cerevisiae evolution, resulting in both arm homogenization and inversion of the IR spacer. These events in turn resulted in nonsynonymous substitutions and up to five neighboring amino acid replacements in a single gene. The study demonstrates that template switching is a powerful generator of multiple substitutions within codons. Additionally, some template switching events occurred more than once during S. cerevisiae evolution. Our findings suggest that template switching constitutes a general mutagenic mechanism that results in both nonsynonymous substitutions and parallel evolution, which are traditionally considered as evidence for positive selection, without the need for adaptive explanations.

Abraxas suppresses DNA end resection and limits break-induced replication by controlling SLX4/MUS81 chromatin loading in response to TOP1 inhibitor-induced DNA damage

Although homologous recombination (HR) is indicated as a high-fidelity repair mechanism, break-induced replication (BIR), a subtype of HR, is a mutagenic mechanism that leads to chromosome rearrangements. It remains poorly understood how cells suppress mutagenic BIR. Trapping of Topoisomerase 1 by camptothecin (CPT) in a cleavage complex on the DNA can be transformed into single-ended double-strand breaks (seDSBs) upon DNA replication or colliding with transcriptional machinery. Here, we demonstrate a role of Abraxas in limiting seDSBs undergoing BIR-dependent mitotic DNA synthesis. Through counteracting K63-linked ubiquitin modification, Abraxas restricts SLX4/Mus81 recruitment to CPT damage sites for cleavage and subsequent resection processed by MRE11 endonuclease, CtIP, and DNA2/BLM. Uncontrolled SLX4/MUS81 loading and excessive end resection due to Abraxas-deficiency leads to increased mitotic DNA synthesis via RAD52- and POLD3- dependent, RAD51-independent BIR and extensive chromosome aberrations. Our work implicates Abraxas/BRCA1-A complex as a critical regulator that restrains BIR for protection of genome stability.

Small-Molecule Antiviral β-D-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance

ABSTRACT Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3′-5′ exoribonuclease (ExoN). β-d-N4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 μM) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 μM) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication. IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, β-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

The mutagenic mechanism of oxygenated alkylhydrazones occurs through alkyl radicals and alkyldiazonium ions

Hydrazone hydroperoxides were formed by autoxidation and their mutagenicity was derived from the alkyldiazonium ion and the radical species.

Improving the Yields of Carotene from Red Yeast by Using Ultra High Pressure and its Mutagenic Mechanism

The R. glutinis NR98 of high-producing β-carotene was treated by ultra high pressure (UHP) of 100~500 MPa for 10~30 min. The survival curve of NR98 treated for 10 min was saddle, which shows that the effect of UHP on this strain maybe was the cumulation of many biology effects. The rate of β-carotene production from mutant G39 (obtained at 300 MPa for 10 min) was increased by 59.87% compared with the initial strain NR98, and its genetic quality was proved to be stable by experiments. The result of restriction fragment length polymorphism analysis suggested that mutant strain G39 was likely to change in nucleic acid level.

Anti-clastogenic effect of b-glucan extracted from barley towards chemically induced DNA damage in rodent cells

b-Glucan (BG) was tested in vitro to determine its potential clastogenic and/or anti-clastogenic activity, and attempts were made to elucidate its possible mechanism of action by using combinations with an inhibitor of DNA polymerase. The study was carried out on cells deficient (CHO-k1) and cells proficient (HTC) in phases I and II enzymes, and the DNA damage was assessed by the chromosomal aberration assay. BG did not show a clastogenic effect, but was anti-clastogenic in both cell lines used, and at all concentrations tested (2.5, 5 and 10 mg/mL) in combination with damage inducing agents (methylmethane sulfonate in cell line CHO-k1, and methylmethane sulfonate or 2-aminoanthracene in cell line HTC). BG also showed a protective effect in the presence of a DNA polymerase b inhibitor (cytosine arabinoside-3-phosphate, Ara-C), demonstrating that BG does not act through an anti-mutagenic mechanism of action involving DNA polymerase b.