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Nature Communications Jan 2021Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide...
Nonribosomal peptide synthetases containing starter condensation domains direct the biosynthesis of nonribosomal lipopeptides, which generally exhibit wide bioactivities. The acyl chain has strong impacts on bioactivity and toxicity, but the lack of an in-depth understanding of starter condensation domain-mediated lipoinitiation limits the bioengineering of NRPSs to obtain novel derivatives with desired acyl chains. Here, we show that the acyl chains of the lipopeptides rhizomide, holrhizin, and glidobactin were modified by engineering the starter condensation domain, suggesting a workable approach to change the acyl chain. Based on the structure of the mutated starter condensation domain of rhizomide biosynthetic enzyme RzmA in complex with octanoyl-CoA and related point mutation experiments, we identify a set of residues responsible for the selectivity of substrate acyl chains and extend the acyl chains from acetyl to palmitoyl. Furthermore, we illustrate three possible conformational states of starter condensation domains during the reaction cycle of the lipoinitiation process. Our studies provide further insights into the mechanism of lipoinitiation and the engineering of nonribosomal peptide synthetases.
Topics: Acylation; Amino Acid Sequence; Lipids; Lipopeptides; Models, Molecular; Peptide Biosynthesis, Nucleic Acid-Independent; Point Mutation; Protein Domains; Protein Engineering; Substrate Specificity
PubMed: 33436600
DOI: 10.1038/s41467-020-20548-8 -
Schizophrenia Bulletin 2001There is a strong genetic component for schizophrenia risk, but it is unclear how the illness is maintained in the population given the significantly reduced fertility... (Review)
Review
There is a strong genetic component for schizophrenia risk, but it is unclear how the illness is maintained in the population given the significantly reduced fertility of those with the disorder. One possibility is that new mutations occur in schizophrenia vulnerability genes. If so, then those with schizophrenia may have older fathers, because advancing paternal age is the major source of new mutations in humans. This review describes several neurodevelopmental disorders that have been associated with de novo mutations in the paternal germ line and reviews data linking increased schizophrenia risk with older fathers. Several genetic mechanisms that could explain this association are proposed, including paternal germ line mutations, trinucleotide repeat expansions, and alterations in genetic imprinting in one or several genes involved in neurodevelopment. Animal models may be useful in exploring these and other explanations for the paternal age effect and they may provide a novel approach for gene identification. Finally, it is proposed that environmental exposures of the father, as well as those of the mother and developing fetus, may be relevant to the etiology of schizophrenia.
Topics: Fathers; Genomic Imprinting; Humans; Male; Point Mutation; Risk Factors; Schizophrenia; Schizophrenic Psychology; Sex Factors
PubMed: 11596842
DOI: 10.1093/oxfordjournals.schbul.a006882 -
Journal of the Neurological Sciences Nov 1998Polymerase chain reaction (PCR) based methods for the diagnosis and screening of the mitochondrial disorders have been well established. A number of tissues are...
Polymerase chain reaction (PCR) based methods for the diagnosis and screening of the mitochondrial disorders have been well established. A number of tissues are routinely used. In this study, we compared the detection rate for MELAS A3243G point mutation in muscle, blood and hair follicles. Ten subjects were studied; mean age was 47 years, (SD 16, range 23-73). All ten subjects had the MELAS A3243G point mutation detected in muscle and hair follicles, but only five had the abnormality in blood samples. The rate of detection of the point mutation in blood samples was age dependent. MtDNA analysis on hair follicles is as sensitive as muscle in detecting this mutation. Analysis using blood samples is not as sensitive, particularly in older subjects. The absence of the mutation in blood samples suggests that there is a preferential selection process for normal (wild type) mtDNA over time. This may be related to the rate of cell division and energy requirements of each tissue.
Topics: Adult; Aged; Aging; Blood Physiological Phenomena; DNA, Mitochondrial; Female; Hair Follicle; Humans; MELAS Syndrome; Male; Middle Aged; Muscles; Point Mutation
PubMed: 9879679
DOI: 10.1016/s0022-510x(98)00179-8 -
Nucleic Acids Research Sep 2018We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and...
We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.
Topics: Animals; Animals, Genetically Modified; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Embryo, Nonmammalian; Gene Editing; Gene Knock-In Techniques; Microinjections; Mutagenesis, Site-Directed; Point Mutation; Zebrafish
PubMed: 29905858
DOI: 10.1093/nar/gky512 -
Annals of the New York Academy of... Apr 2015Drugs that target the folate-synthesis pathway have a long history of effectiveness against a variety of pathogens. As antimalarials, the antifolates were safe and well... (Review)
Review
Drugs that target the folate-synthesis pathway have a long history of effectiveness against a variety of pathogens. As antimalarials, the antifolates were safe and well tolerated, but resistance emerged quickly and has persisted even with decreased drug pressure. The primary determinants of resistance in Plasmodium falciparum are well-described point mutations in the enzymes dihydropteroate synthase and dihydrofolate reductase targeted by the combination sulfadoxine-pyrimethamine. Recent work has highlighted the contributions of additional parasite adaptation to antifolate resistance. In fact, the evolution of antifolate-resistant parasites is multifaceted and complex. Gene amplification of the first enzyme in the parasite folate synthesis pathway, GTP-cyclohydrolase, is strongly associated with resistant parasites and potentially contributes to persistence of resistant parasites. Further understanding of how parasites adjust flux through the folate pathway is important to the further development of alternative agents targeting this crucial synthesis pathway.
Topics: Animals; Antimalarials; Drug Resistance; Folic Acid Antagonists; Humans; Plasmodium falciparum; Point Mutation; Tetrahydrofolate Dehydrogenase
PubMed: 25694157
DOI: 10.1111/nyas.12662 -
Nature Mar 2020Acetaldehyde is a highly reactive, DNA-damaging metabolite that is produced upon alcohol consumption. Impaired detoxification of acetaldehyde is common in the Asian...
Acetaldehyde is a highly reactive, DNA-damaging metabolite that is produced upon alcohol consumption. Impaired detoxification of acetaldehyde is common in the Asian population, and is associated with alcohol-related cancers. Cells are protected against acetaldehyde-induced damage by DNA crosslink repair, which when impaired causes Fanconi anaemia (FA), a disease resulting in failure to produce blood cells and a predisposition to cancer. The combined inactivation of acetaldehyde detoxification and the FA pathway induces mutation, accelerates malignancies and causes the rapid attrition of blood stem cells. However, the nature of the DNA damage induced by acetaldehyde and how this is repaired remains a key question. Here we generate acetaldehyde-induced DNA interstrand crosslinks and determine their repair mechanism in Xenopus egg extracts. We find that two replication-coupled pathways repair these lesions. The first is the FA pathway, which operates using excision-analogous to the mechanism used to repair the interstrand crosslinks caused by the chemotherapeutic agent cisplatin. However, the repair of acetaldehyde-induced crosslinks results in increased mutation frequency and an altered mutational spectrum compared with the repair of cisplatin-induced crosslinks. The second repair mechanism requires replication fork convergence, but does not involve DNA incisions-instead the acetaldehyde crosslink itself is broken. The Y-family DNA polymerase REV1 completes repair of the crosslink, culminating in a distinct mutational spectrum. These results define the repair pathways of DNA interstrand crosslinks caused by an endogenous and alcohol-derived metabolite, and identify an excision-independent mechanism.
Topics: Acetaldehyde; Animals; Cisplatin; Cross-Linking Reagents; DNA; DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Ethanol; Fanconi Anemia; Mutagenesis; Nucleotidyltransferases; Point Mutation; Xenopus; Xenopus Proteins
PubMed: 32132710
DOI: 10.1038/s41586-020-2059-5 -
Journal of Medical Genetics Feb 2017Severe, disease-causing germline mitochondrial (mt)DNA mutations are maternally inherited or arise de novo. Strategies to prevent transmission are generally available,...
BACKGROUND
Severe, disease-causing germline mitochondrial (mt)DNA mutations are maternally inherited or arise de novo. Strategies to prevent transmission are generally available, but depend on recurrence risks, ranging from high/unpredictable for many familial mtDNA point mutations to very low for sporadic, large-scale single mtDNA deletions. Comprehensive data are lacking for de novo mtDNA point mutations, often leading to misconceptions and incorrect counselling regarding recurrence risk and reproductive options. We aim to study the relevance and recurrence risk of apparently de novo mtDNA point mutations.
METHODS
Systematic study of prenatal diagnosis (PND) and recurrence of mtDNA point mutations in families with de novo cases, including new and published data. 'De novo' based on the absence of the mutation in multiple (postmitotic) maternal tissues is preferred, but mutations absent in maternal blood only were also included.
RESULTS
In our series of 105 index patients (33 children and 72 adults) with (likely) pathogenic mtDNA point mutations, the de novo frequency was 24.6%, the majority being paediatric. PND was performed in subsequent pregnancies of mothers of four de novo cases. A fifth mother opted for preimplantation genetic diagnosis because of a coexisting Mendelian genetic disorder. The mtDNA mutation was absent in all four prenatal samples and all 11 oocytes/embryos tested. A literature survey revealed 137 de novo cases, but PND was only performed for 9 (including 1 unpublished) mothers. In one, recurrence occurred in two subsequent pregnancies, presumably due to germline mosaicism.
CONCLUSIONS
De novo mtDNA point mutations are a common cause of mtDNA disease. Recurrence risk is low. This is relevant for genetic counselling, particularly for reproductive options. PND can be offered for reassurance.
Topics: Adult; Child; DNA, Mitochondrial; Female; Genetic Counseling; Genetic Diseases, Inborn; Humans; Male; Maternal Inheritance; Oocytes; Point Mutation; Pregnancy; Preimplantation Diagnosis; Prenatal Diagnosis
PubMed: 27450679
DOI: 10.1136/jmedgenet-2016-103876 -
Nucleic Acids Research Jul 2021Protein-protein interactions play a crucial role in all cellular functions and biological processes and mutations leading to their disruption are enriched in many...
Protein-protein interactions play a crucial role in all cellular functions and biological processes and mutations leading to their disruption are enriched in many diseases. While a number of computational methods to assess the effects of variants on protein-protein binding affinity have been proposed, they are in general limited to the analysis of single point mutations and have been shown to perform poorly on independent test sets. Here, we present mmCSM-PPI, a scalable and effective machine learning model for accurately assessing changes in protein-protein binding affinity caused by single and multiple missense mutations. We expanded our well-established graph-based signatures in order to capture physicochemical and geometrical properties of multiple wild-type residue environments and integrated them with substitution scores and dynamics terms from normal mode analysis. mmCSM-PPI was able to achieve a Pearson's correlation of up to 0.75 (RMSE = 1.64 kcal/mol) under 10-fold cross-validation and 0.70 (RMSE = 2.06 kcal/mol) on a non-redundant blind test, outperforming existing methods. Our method is freely available as a user-friendly and easy-to-use web server and API at http://biosig.unimelb.edu.au/mmcsm_ppi.
Topics: Machine Learning; Mutation, Missense; Point Mutation; Protein Interaction Mapping; Software
PubMed: 33893812
DOI: 10.1093/nar/gkab273 -
Neurobiology of Aging Feb 2016There is a growing body of evidence linking mitochondrial dysfunction, mediated either through inherited mitochondrial DNA (mtDNA) variation or mitochondrial proteomic...
There is a growing body of evidence linking mitochondrial dysfunction, mediated either through inherited mitochondrial DNA (mtDNA) variation or mitochondrial proteomic deficit, to Parkinson's disease (PD). Yet, despite this, the role of somatic mtDNA point mutations and specifically point-mutational burden in PD is poorly understood. Here, we take advantage of recent technical and methodological advances to examine the role of age-related and acquired mtDNA mutation in the largest study of mtDNA in postmortem PD tissue to date. Our data show that PD patients suffer an increase in mtDNA mutational burden in, but no limited to, the substantia nigra pars compacta when compared to matched controls. This mutational burden appears increased in genes encoding cytochrome c oxidase, supportive of previous protein studies of mitochondrial dysfunction in PD. Accepting experimental limitations, our study confirms the important role of age-related mtDNA point mutation in the etiology of PD, moreover, by analyzing 2 distinct brain regions, we are able to show that PD patient brains are more vulnerable to mtDNA mutation overall.
Topics: Cohort Studies; DNA, Mitochondrial; Electron Transport Complex IV; Genetic Association Studies; Humans; Parkinson Disease; Point Mutation
PubMed: 26639157
DOI: 10.1016/j.neurobiolaging.2015.10.036 -
Plant Physiology Jun 2004Most of the genes of an organism are known from sequence, but most of the phenotypes are obscure. Thus, reverse genetics has become an important goal for many... (Review)
Review
Most of the genes of an organism are known from sequence, but most of the phenotypes are obscure. Thus, reverse genetics has become an important goal for many biologists. However, reverse-genetic methodologies are not similarly applicable to all organisms. In the general strategy for reverse genetics that we call TILLING (for Targeting Induced Local Lesions in Genomes), traditional chemical mutagenesis is followed by high-throughput screening for point mutations. TILLING promises to be generally applicable. Furthermore, because TILLING does not involve transgenic modifications, it is attractive not only for functional genomics but also for agricultural applications. Here, we present an overview of the status of TILLING methodology, including Ecotilling, which entails detection of natural variation. We describe public TILLING efforts in Arabidopsis and other organisms, including maize (Zea mays) and zebrafish. We conclude that TILLING, a technology developed in plants, is rapidly being adopted in other systems.
Topics: Agriculture; Animals; Arabidopsis; Genome, Plant; Genomics; Mutagenesis; Point Mutation; Technology Transfer; Zea mays; Zebrafish
PubMed: 15155876
DOI: 10.1104/pp.104.041061