Stochastic organelle genome segregation through Arabidopsis development and reproduction.

Organelle DNA (oDNA) in mitochondria and plastids is vital for plant (and eukaryotic) life. Selection against damaged oDNA is mediated in part by segregation - sorting different oDNA types into different cells in the germline. Plants segregate oDNA very rapidly, with oDNA recombination protein MSH1 a key driver of this segregation, but we have limited knowledge of the dynamics of this segregation within plants and between generations. Here, we reveal how oDNA evolves through Arabidopsis thaliana development and reproduction. We combine stochastic modelling, Bayesian inference, and model selection with new and existing tissue-specific oDNA measurements from heteroplasmic Arabidopsis plant lines through development and between generations. Segregation proceeds gradually but continually during plant development, with a more rapid increase between inflorescence formation and the next generation. When MSH1 is compromised, the majority of observed segregation can be achieved through partitioning at cell divisions. When MSH1 is functional, mtDNA segregation is far more rapid; we show that increased oDNA gene conversion is a plausible mechanism quantitatively explaining this acceleration. These findings reveal the quantitative, time-dependent details of oDNA segregation in Arabidopsis. We also discuss the support for different models of the plant germline provided by these observations.

Molecular dynamics of mismatch detection-How MutS uses indirect readout to find errors in DNA.

The mismatch repair protein MutS safeguards genomic integrity by finding and initiating repair of basepairing errors in DNA. Single-molecule studies show MutS diffusing on DNA, presumably scanning for mispaired/unpaired bases, and crystal structures show a characteristic "mismatch-recognition" complex with DNA enclosed within MutS and kinked at the site of error. But how MutS goes from scanning thousands of Watson-Crick basepairs to recognizing rare mismatches remains unanswered, largely because atomic-resolution data on the search process are lacking. Here, 10 µs all-atom molecular dynamics simulations of Thermus aquaticus MutS bound to homoduplex DNA and T-bulge DNA illuminate the structural dynamics underlying the search mechanism. MutS-DNA interactions constitute a multistep mechanism to check DNA over two helical turns for its 1) shape, through contacts with the sugar-phosphate backbone, 2) conformational flexibility, through bending/unbending engineered by large-scale motions of the clamp domain, and 3) local deformability, through basepair destabilizing contacts. Thus, MutS can localize a potential target by indirect readout due to lower energetic costs of bending mismatched DNA and identify a site that distorts easily due to weaker base stacking and pairing as a mismatch. The MutS signature Phe-X-Glu motif can then lock in the mismatch-recognition complex to initiate repair.

Sensory plastid-associated PsbP DOMAIN-CONTAINING PROTEIN 3 triggers plant growth- and defense-related epigenetic responses.

Sensory plastids are important in plant responses to environmental changes. Previous studies show that MutS HOMOLOG 1 (MSH1) perturbation in sensory plastids induces heritable epigenetic phenotype adjustment. Previously, the PsbP homolog DOMAIN-CONTAINING PROTEIN 3 (PPD3), a protein of unknown function, was postulated to be an interactor with MSH1. This study investigates the relationship of PPD3 with MSH1 and with plant environmental sensing. The ppd3 mutant displays a whole-plant phenotype variably altered in growth rate, flowering time, reactive oxygen species (ROS) modulation and response to salt, with effects on meristem growth. Present in both chloroplasts and sensory plastids, PPD3 colocalized with MSH1 in root tips but not in leaf tissues. The suppression or overexpression of PPD3 affected the plant growth rate and stress tolerance, and led to a heritable, heterogenous 'memory' state with both dwarfed and vigorous growth phenotypes. Gene expression and DNA methylome data sets from PPD3-OX and derived memory states showed enrichment in growth versus defense networks and meristem effects. Our results support a model of sensory plastid influence on nuclear epigenetic behavior and ppd3 as a second trigger, functioning within meristem plastids to recalibrate growth plasticity.

Genome-wide contributions of the MutSα- and MutSß-dependent DNA mismatch repair pathways to the maintenance of genetic stability in Saccharomyces cerevisiae.

The DNA mismatch repair (MMR) system is a major DNA repair system that suppresses both inherited and sporadic cancers in humans. In eukaryotes, the MutSα-dependent and MutSß-dependent MMR pathways correct DNA polymerase errors. Here, we investigated these two pathways on a whole genome level in Saccharomyces cerevisiae. We found that inactivation of MutSα-dependent MMR increases the genome-wide mutation rate by ∼17-fold and loss of MutSß-dependent MMR elevates the genome-wide mutation rate by ∼4-fold. We also found that MutSα-dependent MMR does not show a preference for protecting coding or noncoding DNA from mutations, whereas MutSß-dependent MMR preferentially protects noncoding DNA from mutations. The most frequent mutations in the msh6Δ strain are C>T transitions, whereas 1- to 6-bp deletions are the most common genetic alterations in the msh3Δ strain. Strikingly, MutSα-dependent MMR is more important than MutSß-dependent MMR for protection from 1-bp insertions, while MutSß-dependent MMR has a more critical role in the defense against 1-bp deletions and 2- to 6-bp indels. We also determined that a mutational signature of yeast MSH6 loss is similar to mutational signatures of human MMR deficiency. Furthermore, our analysis showed that compared to other 5'-NCN-3' trinucleotides, 5'-GCA-3' trinucleotides are at the highest risk of accumulating C>T transitions at the central position in the msh6Δ cells and that the presence of a G/A base at the -1 position is important for the efficient MutSα-dependent suppression of C>T transitions. Our results highlight key differences between the roles of the MutSα-dependent and MutSß-dependent MMR pathways.

New DNA Plasmid Model for Studying DNA Mismatch Repair Response to the G4 Structure.

G-quadruplexes (G4s), the most widely studied alternative DNA structures, are implicated in the regulation of the key cellular processes. In recent years, their involvement in DNA repair machinery has become the subject of intense research. Here, we evaluated the effect of G4 on the prokaryotic DNA mismatch repair (MMR) pathway from two bacterial sources with different mismatch repair mechanisms. The G4 folding, which competes with the maintenance of double-stranded DNA, is known to be controlled by numerous opposing factors. To overcome the kinetic barrier of G4 formation, we stabilized a parallel G4 formed by the d(GGGT)4 sequence in a DNA plasmid lacking a fragment complementary to the G4 motif. Unlike commonly used isolated G4 structures, our plasmid with an embedded stable G4 structure contained elements, such as a MutH cleavage site, required to initiate the repair process. G4 formation in the designed construct was confirmed by Taq polymerase stop assay and dimethyl sulfate probing. The G4-carrying plasmid, together with control ones (lacking a looped area or containing unstructured d(GT)8 insert instead of the G4 motif), were used as new type models to answer the question of whether G4 formation interferes with DNA cleavage as a basic function of MMR.

Serial enrichment of heteroduplex DNA using a MutS-magnetic bead system.

Numerous applications in molecular biology and genomics require characterization of mutant DNA molecules present at low levels within a larger sample of non-mutant DNA. This is often achieved either by selectively amplifying mutant DNA, or by sequencing all the DNA followed by computational identification of the mutant DNA. However, selective amplification is challenging for insertions and deletions (indels). Additionally, sequencing all the DNA in a sample may not be cost effective when only the presence of a mutation needs to be ascertained rather than its allelic fraction. The MutS protein evolved to detect DNA heteroduplexes in which the two DNA strands are mismatched. Prior methods have utilized MutS to enrich mutant DNA by hybridizing mutant to non-mutant DNA to create heteroduplexes. However, the purity of heteroduplex DNA these methods achieve is limited because they can only feasibly perform one or two enrichment cycles. We developed a MutS-magnetic bead system that enables rapid serial enrichment cycles. With six cycles, we achieve complete purification of heteroduplex indel DNA originally present at a 5% fraction and over 40-fold enrichment of heteroduplex DNA originally present at a 1% fraction. This system may enable novel approaches for enriching mutant DNA for targeted sequencing.

MutS functions as a clamp loader by positioning MutL on the DNA during mismatch repair.

Highly conserved MutS and MutL homologs operate as protein dimers in mismatch repair (MMR). MutS recognizes mismatched nucleotides forming ATP-bound sliding clamps, which subsequently load MutL sliding clamps that coordinate MMR excision. Several MMR models envision static MutS-MutL complexes bound to mismatched DNA via a positively charged cleft (PCC) located on the MutL N-terminal domains (NTD). We show MutL-DNA binding is undetectable in physiological conditions. Instead, MutS sliding clamps exploit the PCC to position a MutL NTD on the DNA backbone, likely enabling diffusion-mediated wrapping of the remaining MutL domains around the DNA. The resulting MutL sliding clamp enhances MutH endonuclease and UvrD helicase activities on the DNA, which also engage the PCC during strand-specific incision/excision. These MutS clamp-loader progressions are significantly different from the replication clamp-loaders that attach the polymerase processivity factors ß-clamp/PCNA to DNA, highlighting the breadth of mechanisms for stably linking crucial genome maintenance proteins onto DNA.

Long-read sequencing characterizes mitochondrial and plastid genome variants in Arabidopsis msh1 mutants.

The abundant repeats in plant mitochondrial genomes can cause rapid genome rearrangements and are also a major obstacle in short-read sequencing studies. Nuclear-encoded proteins such as MSH1 are known to suppress the generation of repeat-associated mitochondrial genome variants, but our understanding of these mechanisms has been constrained by the limitations of short-read technologies. Here, we used highly accurate long-read sequencing (PacBio HiFi) to characterize mitochondrial and plastid genome variants in Arabidopsis thaliana msh1 mutant individuals. The HiFi reads provided a global view of recombination dynamics with detailed quantification of parental and crossover recombination products for both large and small repeats. We found that recombination breakpoints were distributed relatively evenly across the length of repeated sequences and detected widespread internal exchanges of sequence variants between pairs of imperfect repeats in the mitochondrial genome of msh1 mutants. Long-read assemblies of mitochondrial genomes from seven other A. thaliana wild-type accessions differed by repeat-mediated structural rearrangements similar to those observed in msh1 mutants, but they were all in a simple low-heteroplasmy state. The Arabidopsis plastid genome generally lacks small repeats and exhibited a very different pattern of variant accumulation in msh1 mutants compared with the mitochondrial genome. Our data illustrate the power of HiFi technology in studying repeat-mediated recombination in plant organellar genomes and improved the sequence resolution for recombinational processes suppressed by MSH1. Plant organellar genomes can undergo rapid rearrangements. Long-read sequencing provides a detailed and quantitative view of mitochondrial and plastid genome variants normally suppressed by MSH1, advancing our understanding of plant organellar genome dynamics.

Social networks in the single cell.

This article comments on: Chustecki JM, Etherington RD, Gibbs DJ, Johnston IG. 2022. Altered collective mitochondrial dynamics in the Arabidopsis msh1 mutant compromising organelle DNA maintenance. Journal of Experimental Botany 73, 5428-5439. Plant mitochondrial DNA (mtDNA) can become damaged in many ways. A major repair mechanism is homologous recombination, which requires an undamaged DNA template. Presumably, this template comes from a different mitochondrion in the same cell. Plant mitochondria undergo fission and fusion to form transient networks which could allow the exchange of genetic information. To test this hypothesis, Chustecki et al. (2022) used msh1 mutants with defective DNA repair, and showed that mitochondrial interactions increased, revealing a link between the physical and genetic behavior of mitochondria.

MutS recognition of mismatches within primed DNA replication intermediates.

MutS initiates mismatch repair by recognizing mismatches in newly replicated DNA. Specific interactions between MutS and mismatches within double-stranded DNA promote ADP-ATP exchange and a conformational change into a sliding clamp. Here, we demonstrated that MutS from Pseudomonas aeruginosa associates with primed DNA replication intermediates. The predicted structure of this MutS-DNA complex revealed a new DNA binding site, in which Asn 279 and Arg 272 appeared to directly interact with the 3'-OH terminus of primed DNA. Mutation of these residues resulted in a noticeable defect in the interaction of MutS with primed DNA substrates. Remarkably, MutS interaction with a mismatch within primed DNA induced a compaction of the protein structure and impaired the formation of an ATP-bound sliding clamp. Our findings reveal a novel DNA binding mode, conformational change and intramolecular signaling for MutS recognition of mismatches within primed DNA structures.

Exploiting the distinctive properties of the bacterial and human MutS homolog sliding clamps on mismatched DNA.

MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins. We found that ATP-bound MSH complexes bound to blocked-end or very long mismatched DNAs were extremely stable over a range of ionic conditions. These observations underpinned the development of a high-throughput Förster resonance energy transfer system that specifically detects the formation of MSH sliding clamps on mismatched DNA. The Förster resonance energy transfer system is capable of distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 and appears suitable for chemical inhibitor screens. Taken together, our results provide additional insight into MSH sliding clamps as well as methods to distinguish their functions in mismatch repair.

Methylome decoding of RdDM-mediated reprogramming effects in the Arabidopsis MSH1 system.

BACKGROUND: Plants undergo programmed chromatin changes in response to environment, influencing heritable phenotypic plasticity. The RNA-directed DNA methylation (RdDM) pathway is an essential component of this reprogramming process. The relationship of epigenomic changes to gene networks on a genome-wide basis has been elusive, particularly for intragenic DNA methylation repatterning. RESULTS: Epigenomic reprogramming is tractable to detailed study and cross-species modeling in the MSH1 system, where perturbation of the plant-specific gene MSH1 triggers at least four distinct nongenetic states to impact plant stress response and growth vigor. Within this system, we have defined RdDM target loci toward decoding phenotype-relevant methylome data. We analyze intragenic methylome repatterning associated with phenotype transitions, identifying state-specific cytosine methylation changes in pivotal growth-versus-stress, chromatin remodeling, and RNA spliceosome gene networks that encompass 871 genes. Over 77% of these genes, and 81% of their central network hubs, are functionally confirmed as RdDM targets based on analysis of mutant datasets and sRNA cluster associations. These dcl2/dcl3/dcl4-sensitive gene methylation sites, many present as singular cytosines, reside within identifiable sequence motifs. These data reflect intragenic methylation repatterning that is targeted and amenable to prediction. CONCLUSIONS: A prevailing assumption that biologically relevant DNA methylation variation occurs predominantly in density-defined differentially methylated regions overlooks behavioral features of intragenic, single-site cytosine methylation variation. RdDM-dependent methylation changes within identifiable sequence motifs reveal gene hubs within networks discriminating stress response and growth vigor epigenetic phenotypes. This study uncovers components of a methylome "code" for de novo intragenic methylation repatterning during plant phenotype transitions.

Sorting of mitochondrial and plastid heteroplasmy in Arabidopsis is extremely rapid and depends on MSH1 activity.

The fate of new mitochondrial and plastid mutations depends on their ability to persist and spread among the numerous organellar genome copies within a cell (heteroplasmy). The extent to which heteroplasmies are transmitted across generations or eliminated through genetic bottlenecks is not well understood in plants, in part because their low mutation rates make these variants so infrequent. Disruption of MutS Homolog 1 (MSH1), a gene involved in plant organellar DNA repair, results in numerous de novo point mutations, which we used to quantitatively track the inheritance of single nucleotide variants in mitochondrial and plastid genomes in Arabidopsis. We found that heteroplasmic sorting (the fixation or loss of a variant) was rapid for both organelles, greatly exceeding rates observed in animals. In msh1 mutants, plastid variants sorted faster than those in mitochondria and were typically fixed or lost within a single generation. Effective transmission bottleneck sizes (N) for plastids and mitochondria were N ∼ 1 and 4, respectively. Restoring MSH1 function further increased the rate of heteroplasmic sorting in mitochondria (N ∼ 1.3), potentially because of its hypothesized role in promoting gene conversion as a mechanism of DNA repair, which is expected to homogenize genome copies within a cell. Heteroplasmic sorting also favored GC base pairs. Therefore, recombinational repair and gene conversion in plant organellar genomes can potentially accelerate the elimination of heteroplasmies and bias the outcome of this sorting process.

Altered collective mitochondrial dynamics in the Arabidopsis msh1 mutant compromising organelle DNA maintenance.

Mitochondria form highly dynamic populations in the cells of plants (and almost all eukaryotes). The characteristics and benefits of this collective behaviour, and how it is influenced by nuclear features, remain to be fully elucidated. Here, we use a recently developed quantitative approach to reveal and analyse the physical and collective 'social' dynamics of mitochondria in an Arabidopsis msh1 mutant where the organelle DNA maintenance machinery is compromised. We use a newly created line combining the msh1 mutant with mitochondrially targeted green fluorescent protein (GFP), and characterize mitochondrial dynamics with a combination of single-cell time-lapse microscopy, computational tracking, and network analysis. The collective physical behaviour of msh1 mitochondria is altered from that of the wild type in several ways: mitochondria become less evenly spread, and networks of inter-mitochondrial encounters become more connected, with greater potential efficiency for inter-organelle exchange-reflecting a potential compensatory mechanism for the genetic challenge to the mitochondrial DNA population, supporting more inter-organelle exchange. We find that these changes are similar to those observed in friendly, where mitochondrial dynamics are altered by a physical perturbation, suggesting that this shift to higher connectivity may reflect a general response to mitochondrial challenges, where physical dynamics of mitochondria may be altered to control the genetic structure of the mtDNA population.

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA-protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2'-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA-protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA-protein interactions because of high selectivity of cysteine trapping.

Structural and functional insights into the mechanism by which MutS2 recognizes a DNA junction.

MutS family proteins are classified into MutS-I and -II lineages: MutS-I recognizes mismatched DNA and initiates mismatch repair, whereas MutS-II recognizes DNA junctions to modulate recombination. MutS-I forms dimeric clamp-like structures enclosing the mismatched DNA, and its composite ATPase sites regulate DNA-binding modes. Meanwhile, the structures of MutS-II have not been determined; accordingly, it remains unknown how MutS-II recognizes DNA junctions and how nucleotides control DNA binding. Here, we solved the ligand-free and ADP-bound crystal structures of bacterial MutS2 belonging to MutS-II. MutS2 also formed a dimeric clamp-like structure with composite ATPase sites. The ADP-bound MutS2 was more flexible compared to the ligand-free form and could be more suitable for DNA entry. The inner hole of the MutS2 clamp was two times larger than that of MutS-I, and site-directed mutagenesis analyses revealed DNA-binding sites at the inner hole. Based on these, a model is proposed that describes how MutS2 recognizes DNA junctions.

Large dataset of octocoral mitochondrial genomes provides new insights into mt-mutS evolution and function.

All studied octocoral mitochondrial genomes (mt-genomes) contain a homologue of the Escherichia coli mutS gene, a member of a gene family encoding proteins involved in DNA mismatch repair, other types of DNA repair, meiotic recombination, and other functions. Although mutS homologues are found in all domains of life, as well as viruses, octocoral mt-mutS is the only such gene found in an organellar genome. While the function of mtMutS is not known, its domain architecture, conserved sequence, and presence of several characteristic residues suggest its involvement in mitochondrial DNA repair. This inference is supported by exceptionally low rates of mt-sequence evolution observed in octocorals. Previous studies of mt-mutS have been limited by the small number of octocoral mt-genomes available. We utilized sequence-capture data from the recent Quattrini et al. 2020 study [Nature Ecology & Evolution 4:1531-1538] to assemble complete mt-genomes for 94 species of octocorals. Combined with sequences publicly available in GenBank, this resulted in a dataset of 184 complete mt-genomes, which we used to re-analyze the conservation and evolution of mt-mutS. In our analysis, we discovered the first case of mt-mutS loss among octocorals in one of the two Pseudoanthomastus spp. assembled from Quattrini et al. data. This species displayed accelerated rate and changed patterns of nucleotide substitutions in mt-genome, which we argue provide additional evidence for the role of mtMutS in DNA repair. In addition, we found accelerated mt-sequence evolution in the presence of mt-mutS in several octocoral lineages. This accelerated evolution did not appear to be the result of relaxed selection pressure and did not entail changes in patterns of nucleotide substitutions. Overall, our results support previously reported patterns of conservation in mt-mutS and suggest that mtMutS is involved in DNA repair in octocoral mitochondria. They also indicate that the presence of mt-mutS contributes to, but does not fully explain, the low rates of sequence evolution in octocorals.

Cryogenic electron microscopy structures reveal how ATP and DNA binding in MutS coordinates sequential steps of DNA mismatch repair.

DNA mismatch repair detects and corrects mismatches introduced during DNA replication. The protein MutS scans for mismatches and coordinates the repair cascade. During this process, MutS undergoes multiple conformational changes in response to ATP binding, hydrolysis and release, but how ATP induces the various MutS conformations is incompletely understood. Here we present four cryogenic electron microscopy structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle that reveal how ATP binding and hydrolysis induce closing and opening of the MutS dimer, respectively. Biophysical analysis demonstrates how DNA binding modulates the ATPase cycle by prevention of hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single-stranded DNA that is produced during removal of the daughter strand. The combination of ATP binding and hydrolysis and its modulation by DNA enables MutS to adopt the different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

The methylation-independent mismatch repair machinery in Pseudomonas aeruginosa.

Over the last 70 years, we've all gotten used to an Escherichia coli-centric view of the microbial world. However, genomics, as well as the development of improved tools for genetic manipulation in other species, is showing us that other bugs do things differently, and that we cannot simply extrapolate from E. coli to everything else. A particularly good example of this is encountered when considering the mechanism(s) involved in DNA mismatch repair by the opportunistic human pathogen, Pseudomonas aeruginosa (PA). This is a particularly relevant phenotype to examine in PA, since defects in the mismatch repair (MMR) machinery often give rise to the property of hypermutability. This, in turn, is linked with the vertical acquisition of important pathoadaptive traits in the organism, such as antimicrobial resistance. But it turns out that PA lacks some key genes associated with MMR in E. coli, and a closer inspection of what is known (or can be inferred) about the MMR enzymology reveals profound differences compared with other, well-characterized organisms. Here, we review these differences and comment on their biological implications.