Project description:DNA replication errors are a major driver of evolutionâ??from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The modelâ??Origin-Dependent Inverted-Repeat Amplification (ODIRA)â??proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication errorâ??the ligation of leading and lagging nascent strands to create a "closed" forkâ??can occur in vitro at short, interrupted inverted repeats. The removal of molecules with closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parentâ??a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homolog prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution These are all CGH arrays comparing DNA copy number between evolved yeast strains and a euploid wt strain.

Project description:DNA replication errors are a major driver of evolution—from single nucleotide polymorphisms to large-scale copy number variations (CNVs). Here we test a specific replication-based model to explain the generation of interstitial, inverted triplications. While no genetic information is lost, the novel inversion junctions and increased copy number of the included sequences create the potential for adaptive phenotypes. The model—Origin-Dependent Inverted-Repeat Amplification (ODIRA)—proposes that a replication error at pre-existing short, interrupted, inverted repeats in genomic sequences generates an extrachromosomal, inverted dimeric, autonomously replicating intermediate; subsequent genomic integration of the dimer yields this class of CNV without loss of distal chromosomal sequences. We used a combination of in vitro and in vivo approaches to test the feasibility of the proposed replication error and its downstream consequences on chromosome structure in the yeast Saccharomyces cerevisiae. We show that the proposed replication error—the ligation of leading and lagging nascent strands to create “closed” forks—can occur in vitro at short, interrupted inverted repeats. The removal of molecules with two closed forks results in a hairpin-capped linear duplex that we show replicates in vivo to create an inverted, dimeric plasmid that subsequently integrates into the genome by homologous recombination, creating an inverted triplication. While other models have been proposed to explain inverted triplications and their derivatives, our model can also explain the generation of human, de novo, inverted amplicons that have a 2:1 mixture of sequences from both homologues of a single parent—a feature readily explained by a plasmid intermediate that arises from one homologue and integrates into the other homologue prior to meiosis. Our tests of key features of ODIRA lend support to this mechanism and suggest further avenues of enquiry to unravel the origins of interstitial, inverted CNVs pivotal in human health and evolution.

Project description:Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry. Quantitative comparisons with proteomes of unfractionated proplastids and chloroplasts facilitated the determination of nucleoid-enriched proteins. This nucleoid-enriched proteome included proteins involved in DNA replication, organization, and repair as well as transcription, mRNA processing, splicing, and editing. Many proteins of unknown function, including pentatricopeptide repeat (PPR), tetratricopeptide repeat (TPR), DnaJ, and mitochondrial transcription factor (mTERF) domain proteins, were identified. Strikingly, 70S ribosome and ribosome assembly factors were strongly overrepresented in nucleoid fractions, but protein chaperones were not. Our analysis strongly suggests that mRNA processing, splicing, and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur cotranscriptionally. The plastid developmental state did not dramatically change the nucleoid-enriched proteome but did quantitatively shift the predominating function from RNA metabolism in undeveloped plastids to translation and homeostasis in chloroplasts. This study extends the known maize plastid proteome by hundreds of proteins, including more than 40 PPR and mTERF domain proteins, and provides a resource for targeted studies on plastid gene expression. Details of protein identification and annotation are provided in the Plant Proteome Database.

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Monsonia

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