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G3 (Bethesda, Md.) Aug 2020The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and...
The mutation rate and mutations' effects on fitness are crucial to evolution. Mutation rates are under selection due to linkage between mutation rate modifiers and mutations' effects on fitness. The linkage between a higher mutation rate and more beneficial mutations selects for higher mutation rates, while the linkage between a higher mutation rate and more deleterious mutations selects for lower mutation rates. The net direction of selection on mutations rates depends on the fitness landscape, and a great deal of work has elucidated the fitness landscapes of mutations. However, tests of the effect of varying a mutation rate on evolution in a single organism in a single environment have been difficult. This has been studied using strains of antimutators and mutators, but these strains may differ in additional ways and typically do not allow for continuous variation of the mutation rate. To help investigate the effects of the mutation rate on evolution, we have genetically engineered a strain of with a point mutation rate that can be smoothly varied over two orders of magnitude. We did this by engineering a strain with inducible control of the mismatch repair proteins MutH and MutL. We used this strain in an approximately 350 generation evolution experiment with controlled variation of the mutation rate. We confirmed the construct and the mutation rate were stable over this time. Sequencing evolved strains revealed a higher number of single nucleotide polymorphisms at higher mutations rates, likely due to either the beneficial effects of these mutations or their linkage to beneficial mutations.
Topics: Escherichia coli; Escherichia coli Proteins; Mutation; Mutation Rate; Point Mutation; Selection, Genetic
PubMed: 32503807
DOI: 10.1534/g3.120.401124 -
BMC Bioinformatics Oct 2020Genomic profiling of solid human tumors by projects such as The Cancer Genome Atlas (TCGA) has provided important information regarding the somatic alterations that...
BACKGROUND
Genomic profiling of solid human tumors by projects such as The Cancer Genome Atlas (TCGA) has provided important information regarding the somatic alterations that drive cancer progression and patient survival. Although researchers have successfully leveraged TCGA data to build prognostic models, most efforts have focused on specific cancer types and a targeted set of gene-level predictors. Less is known about the prognostic ability of pathway-level variables in a pan-cancer setting. To address these limitations, we systematically evaluated and compared the prognostic ability of somatic point mutation (SPM) and copy number variation (CNV) data, gene-level and pathway-level models for a diverse set of TCGA cancer types and predictive modeling approaches.
RESULTS
We evaluated gene-level and pathway-level penalized Cox proportional hazards models using SPM and CNV data for 29 different TCGA cohorts. We measured predictive accuracy as the concordance index for predicting survival outcomes. Our comprehensive analysis suggests that the use of pathway-level predictors did not offer superior predictive power relative to gene-level models for all cancer types but had the advantages of robustness and parsimony. We identified a set of cohorts for which somatic alterations could not predict prognosis, and a unique cohort LGG, for which SPM data was more predictive than CNV data and the predictive accuracy is good for all model types. We found that the pathway-level predictors provide superior interpretative value and that there is often a serious collinearity issue for the gene-level models while pathway-level models avoided this issue.
CONCLUSION
Our comprehensive analysis suggests that when using somatic alterations data for cancer prognosis prediction, pathway-level models are more interpretable, stable and parsimonious compared to gene-level models. Pathway-level models also avoid the issue of collinearity, which can be serious for gene-level somatic alterations. The prognostic power of somatic alterations is highly variable across different cancer types and we have identified a set of cohorts for which somatic alterations could not predict prognosis. In general, CNV data predicts prognosis better than SPM data with the exception of the LGG cohort.
Topics: DNA Copy Number Variations; Humans; Point Mutation; Prognosis
PubMed: 33081688
DOI: 10.1186/s12859-020-03791-0 -
Medecine Sciences : M/S Oct 2022An ingenious system for generating thousands of point mutations in yeast genes and measuring their effect on fitness shows convincingly that, for the chosen subset of...
An ingenious system for generating thousands of point mutations in yeast genes and measuring their effect on fitness shows convincingly that, for the chosen subset of representative non-essential genes, silent mutations have as much effect on fitness as missense mutations. In other words, silent mutations are not neutral, at least under these conditions. This result has important implications for evolutionary biology.
Topics: Biological Evolution; Humans; Mutation; Mutation, Missense; Point Mutation
PubMed: 36219087
DOI: 10.1051/medsci/2022126 -
Scientific Reports Sep 2016The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of...
The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.
Topics: Alleles; Base Sequence; CRISPR-Cas Systems; Clone Cells; DNA Repair; DNA, Single-Stranded; Gene Editing; HCT116 Cells; Humans; K562 Cells; Point Mutation; Templates, Genetic; beta-Globins
PubMed: 27609304
DOI: 10.1038/srep32681 -
BMC Biotechnology Feb 2017Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory.
BACKGROUND
Escherichia coli suffer from osmotic stress during succinic acid (SA) production, which reduces the performance of this microbial factory.
RESULTS
Here, we report that a point mutation leading to a single amino acid change (D654Y) within the β-subunit of DNA-dependent RNA polymerase (RpoB) significantly improved the osmotolerance of E. coli. Importation of the D654Y mutation of RpoB into the parental strain, Suc-T110, increased cell growth and SA production by more than 40% compared to that of the control under high glucose osmolality. The transcriptome profile, determined by RNA-sequencing, showed two distinct stress responses elicited by the mutated RpoB that counterbalanced the osmotic stress. Under non-stressed conditions, genes involved in the synthesis and transport of compatible solutes such as glycine-betaine, glutamate or proline were upregulated even without osmotic stimulation, suggesting a "pre-defense" mechanism maybe formed in the rpoB mutant. Under osmotic stressed conditions, genes encoding diverse sugar transporters, which should be down-regulated in the presence of high osmotic pressure, were derepressed in the rpoB mutant. Additional genetic experiments showed that enhancing the expression of the mal regulon, especially for genes that encode the glycoporin LamB and maltose transporter, contributed to the osmotolerance phenotype.
CONCLUSIONS
The D654Y single amino acid substitution in RpoB rendered E. coli cells resistant to osmotic stress, probably due to improved cell growth and viability via enhanced sugar uptake under stressed conditions, and activated a potential "pre-defense" mechanism under non-stressed conditions. The findings of this work will be useful for bacterial host improvement to enhance its resistance to osmotic stress and facilitate bio-based organic acids production.
Topics: DNA-Directed RNA Polymerases; Escherichia coli; Escherichia coli Proteins; Mutagenesis, Site-Directed; Osmotic Pressure; Point Mutation; Stress, Physiological; Succinic Acid; Up-Regulation
PubMed: 28193207
DOI: 10.1186/s12896-017-0337-6 -
Nucleic Acids Research Jul 2023Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite...
Understanding the effects of mutations on protein stability is crucial for variant interpretation and prioritisation, protein engineering, and biotechnology. Despite significant efforts, community assessments of predictive tools have highlighted ongoing limitations, including computational time, low predictive power, and biased predictions towards destabilising mutations. To fill this gap, we developed DDMut, a fast and accurate siamese network to predict changes in Gibbs Free Energy upon single and multiple point mutations, leveraging both forward and hypothetical reverse mutations to account for model anti-symmetry. Deep learning models were built by integrating graph-based representations of the localised 3D environment, with convolutional layers and transformer encoders. This combination better captured the distance patterns between atoms by extracting both short-range and long-range interactions. DDMut achieved Pearson's correlations of up to 0.70 (RMSE: 1.37 kcal/mol) on single point mutations, and 0.70 (RMSE: 1.84 kcal/mol) on double/triple mutants, outperforming most available methods across non-redundant blind test sets. Importantly, DDMut was highly scalable and demonstrated anti-symmetric performance on both destabilising and stabilising mutations. We believe DDMut will be a useful platform to better understand the functional consequences of mutations, and guide rational protein engineering. DDMut is freely available as a web server and API at https://biosig.lab.uq.edu.au/ddmut.
Topics: Deep Learning; Mutation; Point Mutation; Protein Stability; Software; Proteins
PubMed: 37283042
DOI: 10.1093/nar/gkad472 -
Homology directed correction, a new pathway model for point mutation repair catalyzed by CRISPR-Cas.Scientific Reports May 2022Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant...
Gene correction is often referred to as the gold standard for precise gene editing and while CRISPR-Cas systems continue to expand the toolbox for clinically relevant genetic repair, mechanistic hurdles still hinder widespread implementation. One of the most prominent challenges to precise CRISPR-directed point mutation repair centers on the prevalence of on-site mutagenesis, wherein insertions and deletions appear at the targeted site following correction. Here, we introduce a pathway model for Homology Directed Correction, specifically point mutation repair, which enables a foundational analysis of genetic tools and factors influencing precise gene editing. To do this, we modified an in vitro gene editing system which utilizes a cell-free extract, CRISPR-Cas RNP and donor DNA template to catalyze point mutation repair. We successfully direct correction of four unique point mutations which include two unique nucleotide mutations at two separate targeted sites and visualize the repair profiles resulting from these reactions. This extension of the cell-free gene editing system to model point mutation repair may provide insight for understanding the factors influencing precise point mutation correction.
Topics: CRISPR-Cas Systems; Catalysis; Gene Editing; Mutagenesis; Mutation; Point Mutation
PubMed: 35581233
DOI: 10.1038/s41598-022-11808-2 -
Genome Biology Jun 2020Repeat-induced point (RIP) mutation in Neurospora crassa degrades transposable elements by targeting repeats with C→T mutations. Whether RIP affects core genomic...
BACKGROUND
Repeat-induced point (RIP) mutation in Neurospora crassa degrades transposable elements by targeting repeats with C→T mutations. Whether RIP affects core genomic sequence in important ways is unknown.
RESULTS
By parent-offspring whole genome sequencing, we estimate a mutation rate (3.38 × 10 per bp per generation) that is two orders of magnitude higher than reported for any non-viral organism, with 93-98% of mutations being RIP-associated. RIP mutations are, however, relatively rare in coding sequence, in part because RIP preferentially attacks GC-poor long duplicates that interact in three dimensional space, while coding sequence duplicates are rare, GC-rich, short, and tend not to interact. Despite this, with over 5 coding sequence mutations per genome per generation, the mutational burden is an order of magnitude higher than the previously highest observed. Unexpectedly, the majority of these coding sequence mutations appear not to be the direct product of RIP being mostly in non-duplicate sequence and predominantly not C→T mutations. Nonetheless, RIP-deficient strains have over an order of magnitude fewer coding sequence mutations outside of duplicated domains than RIP-proficient strains.
CONCLUSIONS
Neurospora crassa has the highest mutation rate and mutational burden of any non-viral life. While the high rate is largely due to the action of RIP, the mutational burden appears to be RIP-associated but not directly caused by RIP.
Topics: DNA, Fungal; Mutation Rate; Neurospora crassa; Point Mutation; Repetitive Sequences, Nucleic Acid
PubMed: 32546205
DOI: 10.1186/s13059-020-02060-w -
Aging Jun 2021
Topics: Extracellular Vesicles; Gastrointestinal Microbiome; Gene Editing; Humans; Molecular Targeted Therapy; Point Mutation; Progeria
PubMed: 34176790
DOI: 10.18632/aging.203254 -
BMC Medical Genomics Jan 2021In addition to ovarian and breast cancers, loss-of-function mutations in BRCA1 and BRCA2 genes are also linked to an increased risk of pancreatic cancer, with ~ 4 to...
BACKGROUND
In addition to ovarian and breast cancers, loss-of-function mutations in BRCA1 and BRCA2 genes are also linked to an increased risk of pancreatic cancer, with ~ 4 to 7% of pancreatic cancer patients harboring germline BRCA mutations. Most BRCA alterations in pancreatic cancer are frame-shifting indels, stop-gain, and splice-site mutations, but single nucleotide substitutions are rare. Recent studies demonstrated a significant progression-free survival (PFS) benefit from maintenance olaparib, a poly (ADP-ribose) polymerase (PARP) inhibitor administered to patients with germline BRCA mutations and metastatic pancreatic cancer.
CASE PRESENTATION
Here, we report a metastatic pancreatic cancer case who harbored a novel somatic BRCA2 c.6944T > C (p. I2315T) point mutation. After 6 weeks first-line chemotherapy, the patient was refractory to treatment and had a progressive disease. Due to the novel nonsynonymous BRCA2 point mutation, we decided to change the strategy by administering olaparib. The patient benefited from olaparib therapy and achieved a PFS of ~ 6.5 months.
CONCLUSIONS
We describe a patient carrying a novel somatic BRCA2 p. I2315T point mutation, which is first reported in metastatic pancreatic cancer. This case report indicates that a gene mutation-based strategy should be considered in the clinic to provide more effective treatment.
Topics: Female; Genes, BRCA2; Humans; Pancreatic Neoplasms; Phthalazines; Piperazines; Point Mutation
PubMed: 33407459
DOI: 10.1186/s12920-020-00850-6