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At least 19 recordsLinked to original sources
PubMed1977

Analysis of the role of recombination and repair in mutagenesis of Escherichia coli by UV irradiation.

Multiple mutant strains have been tested or their mimicry of the UV-mutagenesis deficiency of a recA single mutant. Revertants to histidine prototrophy and clear plaque mutants of lambda were scored to determine capacity for UV-mutagenesis. Nearly normal capacity was shown by a uvr+ recB- recF- strain, which shows almost no recA-dependent recombination, by uvr- recB+ recF- strains, which show almost no recA-dependent repair and by a uvrA- recB- recF- strain, which shows neither recA-dependent recombination nor repair. Since the uvr mutants can be assumed to show additionally no excision repair, these results may mean that UV-mutagenesis occurs during processes other than recombination and repair. Alternative hypotheses are discussed. The slight difference in mutagenic capacity was traced to the recF single mutation, which blocks the production of unmixed bursts of clear-plaque lambda mutants. Since this accounts for only about 10% of the mutations leading to clear-plaque mutants, it is suggested that there is more than one UV-mutagenic process.

Coliphages↗
PubMed2025

Approaches to Study Proteins Encoded by Essential Genes.

Although the phenotypes and functions of nonessential proteins can be studied by deletion of their coding sequences (both gene copies in diploid organisms), essential genes cannot be deleted unless loss of the encoded protein can be bypassed. Bypass is often achieved by supplementation with the product of the enzyme. However, supplementation cannot bypass loss of essential genes such as those encoding enzymes of DNA or RNA synthesis. To study proteins encoded by essential genes that cannot be bypassed, the mutations must be conditional in nature. The mutant cells must be able to grow under a permissive condition, but fail to grow under a different condition, the nonpermissive condition. Several methods have been developed to obtain conditional mutations in essential genes. Mutations that result in proteins abnormally sensitive to high temperatures are called temperature-sensitive (Ts) mutants and are a widely used type of conditional mutation. An alternative to Ts mutants is the "degron" system to target proteins for destruction by cellular proteases. Approaches to conditionally control the functions of proteins encoded by essential genes, plus the advantages and disadvantages of these and other approaches, will be considered.

Genes, Essential↗
PubMed2025

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

Schizosaccharomyces↗
PubMed2026

Powering Genome Editing in Rice by Harnessing Promising Gene Resources: A Comprehensive Roadmap.

The imprecise breeding methods including recombination breeding, physical/chemical mutagenesis, and marker-assisted breeding have been extensively utilized for trait improvement of rice crop. Despite tremendous progress made through these breeding methods, the critical issues, such as linkage drag, unintended phenotype, and longer duration of time required to breed a cultivar, have been the major limitations. Among the new breeding technologies, genome editing (GE) has become the most promising approach because of its specificity, precision, and speed. Despite its transformative potential, genome editing continues to face several limitations in crop improvement. These include well-recognized policy challenges, such as biosafety regulations and intellectual property constraints, alongside technical barriers like inefficient tissue culture and transformation systems. Additionally, researchers remain constrained by the limited availability of precise gene information necessary for accurate targeted editing and effective trait enhancement. This review presents an analysis of genes that regulate abiotic and biotic stresses, yield, grain quality and nutrition, plant architecture, nutrient absorption and use efficiency, and other agronomically important traits of rice. The trait-wise probable target genes for genome editing have been discussed in detail. This review will serve as a ready reckoner for rice researchers and funding agencies.

Oryza↗
PubMed2026

Prime assembly with linear DNA donors enables large genomic insertions.

Targeted insertion of large DNA fragments has promising applications for genome engineering and gene therapy1,2. Twin prime-editing guide RNAs have enabled relatively large insertions, but the efficiency remains low for insertions greater than 400 base pairs3-6. Here we describe a prime assembly (PA) approach for the insertion of large DNA donor fragments, of which the ends are designed to overlap with the flaps generated by twin prime editing (twinPE). We used PA to insert one or multiple overlapping DNA fragments, with total insertion sizes ranging from 0.1 kb to 11 kb. An inhibitor of non-homologous end joining enhanced both the efficiency and precision of insertions. PA relies on DNA templates that are easily produced, does not require co-delivery of exogenous DNA-dependent DNA polymerases and proceeds in non-cycling cells, suggesting independence from canonical homology-directed repair pathways. Our study demonstrates that PA can initiate Gibson-like assembly in cells to generate gene insertions without double-stranded DNA breaks, recombinases or homology-directed repair.

Animals↗
PubMed2004

Regulation of cell cycle-specific gene expression through cyclin-dependent kinase-mediated phosphorylation of the forkhead transcription factor Fkh2p.

The forkhead transcription factor Fkh2p acts in a DNA-bound complex with Mcm1p and the coactivator Ndd1p to regulate cell cycle-dependent expression of the CLB2 gene cluster in Saccharomyces cerevisiae. Here, we demonstrate that Fkh2p is a target of cyclin-dependent protein kinases and that phosphorylation of Fkh2p promotes interactions between Fkh2p and the coactivator Ndd1p. These phosphorylation-dependent changes in the Fkh2p-Ndd1p complex play an important role in the cell cycle-regulated expression of the CLB2 cluster. Our data therefore identify an important regulatory target for cyclin-dependent kinases in the cell cycle and further our molecular understanding of the key cell cycle regulatory transcription factor Fkh2p.

Binding Sites↗
PubMed2026

Mobile elements in pituitary neuroendocrine tumors: integrative evidence and future directions.

Mobile genetic elements (MGEs), including LINE-1 retrotransposons, Alu and SVA elements, and human endogenous retroviruses (HERVs), constitute nearly half of the human genome and are increasingly understood to influence multiple dimensions of cancer evolution. Yet, pituitary neuroendocrine tumors (PitNETs) remain almost absent from mobilome research, despite exhibiting genomic and epigenetic contexts permissive to retroelement activation. In this review, we synthesize current evidence linking MGEs to PitNET biology and delineate unresolved but testable mechanisms. Structural genomic studies demonstrate that Alu-mediated non-allelic homologous recombination contributes to germline mutagenesis in MEN1 and AIP, reinforcing the notion that repetitive DNA architecture shapes PitNET predisposition. Transcriptomic analyses reveal global derepression of transposable elements and LINE-1 hypomethylation in subsets of tumors, while mechanistic connections to chromatin instability emerge from recurrent ATRX/DAXX deficiency and TP53 inactivation, both established repressors of retroelements. Furthermore, the retrocopy-derived long non-coding RNA RPSAP52 exemplifies how mobilome-origin transcripts can be co-opted as oncogenic regulators in PitNETs, acting through HMGA2-dependent proliferative networks. Preliminary data also suggest endogenous retroviral activation, with consistent upregulation of HERV envelope genes across distinct tumor subtypes. Nevertheless, no study has yet systematically mapped somatic mobile-element insertions (MEIs), quantified LINE-1 protein activity, or profiled HERV expression at locus resolution in PitNETs. Mobilome biology represents a tractable and conceptually rich frontier with diagnostic, prognostic, and therapeutic potential in pituitary tumorigenesis.

Humans↗
PubMedDate not supplied

[Production of temperature-sensitive mutants of vaccinia virus with the aid of nitrosomethylurea, and their preliminary characteristics].

The possibility of using nitrosomethylurea for production of one-hit temperature-sensitive (ts) mutants of vaccinia virus suitable for genetic and biochemical studies was explored. The effect of nitrosomethylurea on replicating DNA of the virus produced 26 ts mutants, the effectiveness of mutagenesis being 10%. In recombination experiments with 12 of them it was shown that all the mutants under study had single ts defects of the genome. All ts mutants were capable of synthesizing DNA under nonpermissive conditions; 16 ts mutants had thermolabile virions.

Animals↗
PubMed2026

A genetic manipulation tool based on the GP35 recombinase for targeted gene editing in mycoplasmas of ruminants.

Pathogenic ruminant mycoplasmas are major etiological agents in cattle and small ruminants and are responsible for substantial economic losses in the livestock industry. Progress in pathogenesis research and vaccine development has been hampered by a lack of effective genetic tools. The applicability of common genome editing platforms, such as CRISPR, is inherently restricted in these organisms owing to their minimal genomes, the absence of a cell wall, and low homologous recombination efficiency. Although transposon-mediated random mutagenesis and single-base editing are currently used in the editing of bovine mycoplasma, the stochastic nature of transposons, the risk of single-base random deamination, and limitations in editing window selection hinder the genetic manipulation of bovine mycoplasma. Here, we introduce a plasmid-based methodology that employs the GP35 recombinase from bacteriophage SPP1 to mediate long single-stranded DNA (ssDNA) recombineering, thereby enabling precise gene insertions and deletions in Mycoplasma bovis, with a positive-editing rate of 77.78% - 100%. This targeted system eliminates the risk of random deamination. Leveraging this tool, we generated a panel of M. bovis mutants affecting metabolic and virulence genes and obtained key insights into Mb0564, identified as a novel adhesin. The 192 to 287 aa region of GP35 is critical for interaction with SSB. Structural conservation analysis further suggested that this GP35-ssDNA editing system possesses a high potential for translation to other ruminant pathogens. Collectively, our approach expands the existing genetic toolkit for M. bovis, advances synthetic biology and M. bovis pathobiology, facilitates vaccine development, and strengthens the control of high-impact livestock diseases in line with the One Health framework.

Animals↗
PubMed2026

MRE11 suppresses germline mutagenesis at meiotic double-strand breaks in mice.

SPO11 forms hundreds of double-strand breaks (DSBs) to initiate meiotic recombination that is normally error-free. However, SPO11 activity can be mutagenic when one chromatid incurs closely spaced DSBs (double cuts), especially when DSBs are dysregulated by loss of the ATM kinase. De novo indels and structural variants can arise via end joining at double cuts within a single hotspot (microdeletions) or at adjacent hotspots separated by at least 30 kb, as we now show, sometimes accompanied by ectopic insertions of double-cut fragments. Here, we investigate how meiotic DSB end processing influences end joining. In MRE11-deficient mouse spermatocytes, which do not resect their DSBs, deletions at double cuts occur readily, with end-joining breakpoint profiles closely matching SPO11 DSB profiles. Microdeletions suggest that two DSBs can be as close as ∼21 bp. The tyrosyl-DNA phosphodiesterase TDP2 contributes to both deletion formation and ectopic insertion of double-cut fragments, presumably by removing SPO11 from DNA ends prior to joining. Finally, observations suggest a cooperative role for MRE11 and ATM in locally regulating DSB distributions. Our findings provide insight into the mechanism of de novo mutation origin, emphasizing the role of meiotic DSBs in shaping genome evolution.

Animals↗
PubMed1978

Recombinational repair of alkylation lesions in phage T4. II. Ethyl methanesulfonate.

Treatment of bacteriophage T4 by ethyl methanesulfonate (EMS) caused more than a doubling in recombination between two rII markers. The functions of genes 47, 46, 32, 30, uvsX and y are known to be required for genetic recombination, and mutants defective in these genes were found to be more sensitive to inactivation by EMS than wild-type phage. This suggests that a recombinational pathway involving the products of these genes may be employed in repairing EMS induced lethal lesions. Genes 45 and denV are apparently not involved in recombination, and mutants defective in these genes were not EMS-sensitive. Gene 47, 46 and y mutants which were defective in the repair of EMS induced lethal lesions had no detectable deficiency in their ability to undergo EMS-induced mutation. This implies that recombinational repair of EMS lesions does not contribute substantially to EMS mutagenesis. The results obtained here with EMS are general similar to the results reported in the preceding paper with MNNG, suggesting that the lesions caused by both of these monofunctional alkylating agents may be eliminated by similar recombinational repair processes.

Alkylation↗
PubMed2026

Integrated signatures define mutational processes in prostate cancer.

Prostate cancer follows a long and heterogeneous disease course with incompletely understood aetiology1. Here we dissect the mutational processes shaping the genomes of 959 donors from the Pan Prostate Cancer Group and assess their clinical relevance. By integrating de novo extracted single-base substitution, insertion-deletion and copy-number signatures with six novel complex structural variant signatures, we identify eight integrated mutational footprints (IMFs) that collectively explain the mutational processes in 85% of primary prostate cancer genomes. IMFs were strongly influenced by regional biases in the genome, most prevalently androgen receptor-mediated mutagenesis and replication stress. Four IMFs, present in 37% of primary tumours, were significantly associated with shorter time to metastasis. These included reactive oxygen-species-driven mutagenesis and both canonical and non-canonical homologous recombination deficiency, the latter being enriched in patients of African ancestry. Extending to the metastatic setting, we found that IMFs predicted sensitivity to androgen receptor pathway inhibitors. Taken together, our study delineates the aetiologies and mutational processes that drive the genomic and clinical heterogeneity of prostate cancer, introduces IMFs as a unifying framework, and highlights their potential to improve both risk stratification and biomarker-guided treatment selection.

Journal Article↗
PubMed1976

Effects of growth temperature and caffeine on genetic responses of Candida albicans to ethyl methanesulfonate, nitrous acid and ultraviolet radiation.

Ultraviolet radiation is more effective than either ethyl methanesulfonate or nitrous acid in inducing reverse mutation from auxotrophy to prototrophy in C. albicans. The killing effect of each of the mutagens is greater for cells grown at 37 C than at 25 C after treatment; mutation frequencies are unaffected by post-treatment growth temperatures. Though caffeine depresses survival of mutagen treated cells at both 25 C or 37 C, its effect is more pronounced at 37 C. Caffeine has no effect on mutagenesis by nitrous acid or ethyl methanesulfonate; it depresses UV mutagenesis, but only at 37 C and at high UV dosages. These findings indicate that UV mutagenesis in C. albicans is mediated by a caffeine-sensitive, recombinational system for DNA repair analogous to those known to occur in other species of yeasts. The repair system of C. albicans is unique in being susceptible to caffeine only at high temperature and when the number of DNA lesions to be repaired is large. The caffeine-sensitive steps in repair critical to UV mutagenesis are not involved in fixing mutations induced by the chemical mutagens tested.

Amino Acids↗
PubMed1979

Uvm mutants of Escherichia coli K12 deficient in UV mutagenesis. II. Further evidence for a novel function in error-prone repair.

Uvm mutants of Escherichia coli K12 selected for defective UV reversion induction have previously been reported to differ considerably from the UV-reversion-less recA and lexA mutants with regard to survival or mutagenic response to UV, X-rays and alkylating agents. In the present study, the phenotypic characterization of uvm mutants was extended to investigate several cellular processes which also may be related to or involved in UV mutagenesis. Like recA and lexA mutations, the uvm mutations exhibit highly reduced Weigle reactivation and normal host cell reactivation of UV irradiated phage lambda. But unlike recA and lexA, the uvm mutations do not impair genetic recombination, UV induction of prophage lambda or R plasmid-mediated UV resistance and mutagenesis. These phenotypical characteristics and preliminary results of genetic mapping lend further support to the assumption that the uvm site may be a novel locus affecting, apart from the recA and lexA loci, the error-prone repair pathway in E. coli.

Bacteriophage lambda↗
PubMed1978

The role of pre-replication and post-replication processes in mutation induction in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine.

Studies were carried out on the repair and fixation of premutational damage induced in Haemophilus influenzae by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The studies employed a temperature-sensitive DNA elongation mutant (dna9) and its combinations with mutants defective in pyrimidine dimer excision (uvr1, uvr2) and in recombination (rec1). The dna9 mutant is shown to be leaky, allowing about 1% of the normal rate of DNA synthesis at the restrictive temperature. Repair of premutational lesions was detected by a decline in mutation frequency with increasing delay in DNA replication in dna9 at the restrictive temperature. This repair is unaffected by the pyrimidine dimer excision system. Mutation fixation was detected by the ability of DNA from treated and then lysed cells to transfer mutants to recipient cells by transformation. Some fixation occurred at the restrictive temperature but much less than at the non-restrictive temperature suggesting that an appreciable minority of the mutations resulted from lesions introduced near the replication fork but that the majority of mutations arise from lesions introduced at some distance from the fork, perhaps randomly. The DNA synthesized immediately after MNNG treatment is of lower molecular weight than normal and returns to normal with time. This return is blocked in the rec1 mutant, suggesting that recombination is involved. The possible role of this process in MNNG mutagenesis is discussed.

DNA Repair↗
PubMed1977

Temperature-sensitive respiratory-deficient mitochondrial mutations: isolation and genetic mapping.

In order to find new genetic loci and functions on the yeast mitochondrial DNA, especially mutations affecting the mitochondrial protein synthesis apparatus, temperature sensitive mutants have been isolated after MnCl2 mutagenesis and mitochondrial and nuclear mutants classified according to their pattern of recombination with three rho- tester strains. Eighteen cold- and heat-sensitive respiratory deficient mitochondrial mutants have been isolated and localized on the mitochondrial genome by deletion mapping using 113 rho- strains. Eight of them appear to represent new loci, among which some are probably mutations of the tRNA and rRNA genes.

Cold Temperature↗
PubMed2026

LINE-1 insertion intermediates recombine with one another or with DNA breaks to form genome rearrangements.

LINE-1 (L1) retrotransposition is common in human cancers and rearrangements at insertion sites can contribute to cancer-driving oncogene amplifications and promote genome instability. However, the mechanisms underlying rearrangements of L1 retrotransposition intermediates are poorly understood. To address this gap, we developed GFP-based recombination reporter assays to study the formation of L1 retrotransposition-mediated rearrangements. Using these reporters combined with long-read sequencing, we find that L1 retrotransposition cDNA intermediates can recombine with distal DNA breaks to generate chromosomal rearrangements. We also find that two independent L1 insertion cDNA intermediates on distinct genomic loci can recombine with each other to generate chromosomal rearrangements. Both types of rearrangements depend on L1-encoded ORF2p endonuclease and reverse transcriptase activities. Using these reporters, we discover that L1 retrotransposition-mediated rearrangements are robustly induced when the recombining sequences share extensive homology and that their formation requires the homologous recombination factor BRCA1. In contrast, we find L1 retrotransposition-mediated rearrangements are suppressed by the mismatch repair factor MSH2 when the recombining sequences contain mismatches. Given the repetitive nature of our genome, these findings highlight the risk of L1 insertion intermediates becoming substrates for aberrant recombination and promoting genome instability.

Long Interspersed Nucleotide Elements↗