MSH1-induced heritable enhanced growth vigor through grafting is associated with the RdDM pathway in plants.

Plants transmit signals long distances, as evidenced in grafting experiments that create distinct rootstock-scion junctions. Noncoding small RNA is a signaling molecule that is graft transmissible, participating in RNA-directed DNA methylation; but the meiotic transmissibility of graft-mediated epigenetic changes remains unclear. Here, we exploit the MSH1 system in Arabidopsis and tomato to introduce rootstock epigenetic variation to grafting experiments. Introducing mutations dcl2, dcl3 and dcl4 to the msh1 rootstock disrupts siRNA production and reveals RdDM targets of methylation repatterning. Progeny from grafting experiments show enhanced growth vigor relative to controls. This heritable enhancement-through-grafting phenotype is RdDM-dependent, involving 1380 differentially methylated genes, many within auxin-related gene pathways. Growth vigor is associated with robust root growth of msh1 graft progeny, a phenotype associated with auxin transport based on inhibitor assays. Large-scale field experiments show msh1 grafting effects on tomato plant performance, heritable over five generations, demonstrating the agricultural potential of epigenetic variation.

Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement.

We report that long double-stranded DNA confined to quasi-1D nanochannels undergoes superdiffusive motion under the action of the enzyme T4 DNA ligase in the presence of necessary co-factors. Inside the confined environment of the nanochannel, double-stranded DNA molecules stretch out due to self-avoiding interactions. In absence of a catalytically active enzyme, we see classical diffusion of the center of mass. However, cooperative interactions of proteins with the DNA can lead to directed motion of DNA molecules inside the nanochannel. Here we show directed motion in this configuration for three different proteins (T4 DNA ligase, MutS, E. coli DNA ligase) in the presence of their energetic co-factors (ATP, NAD+).

Long-Range Signaling in MutS and MSH Homologs via Switching of Dynamic Communication Pathways.

Allostery is conformation regulation by propagating a signal from one site to another distal site. This study focuses on the long-range communication in DNA mismatch repair proteins MutS and its homologs where intramolecular signaling has to travel over 70 Å to couple lesion detection to ATPase activity and eventual downstream repair. Using dynamic network analysis based on extensive molecular dynamics simulations, multiple preserved communication pathways were identified that would allow such long-range signaling. The pathways appear to depend on the nucleotides bound to the ATPase domain as well as the type of DNA substrate consistent with previously proposed functional cycles of mismatch recognition and repair initiation by MutS and homologs. A mechanism is proposed where pathways are switched without major conformational rearrangements allowing for efficient long-range signaling and allostery.

Single-molecule motions and interactions in live cells reveal target search dynamics in mismatch repair.

MutS is responsible for initiating the correction of DNA replication errors. To understand how MutS searches for and identifies rare base-pair mismatches, we characterized the dynamic movement of MutS and the replisome in real time using superresolution microscopy and single-molecule tracking in living cells. We report that MutS dynamics are heterogeneous in cells, with one MutS population exploring the nucleoid rapidly, while another MutS population moves to and transiently dwells at the replisome region, even in the absence of appreciable mismatch formation. Analysis of MutS motion shows that the speed of MutS is correlated with its separation distance from the replisome and that MutS motion slows when it enters the replisome region. We also show that mismatch detection increases MutS speed, supporting the model for MutS sliding clamp formation after mismatch recognition. Using variants of MutS and the replication processivity clamp to impair mismatch repair, we find that MutS dynamically moves to and from the replisome before mismatch binding to scan for errors. Furthermore, a block to DNA synthesis shows that MutS is only capable of binding mismatches near the replisome. It is well-established that MutS engages in an ATPase cycle, which is necessary for signaling downstream events. We show that a variant of MutS with a nucleotide binding defect is no longer capable of dynamic movement to and from the replisome, showing that proper nucleotide binding is critical for MutS to localize to the replisome in vivo. Our results provide mechanistic insight into the trafficking and movement of MutS in live cells as it searches for mismatches.

Mutsß generates both expansions and contractions in a mouse model of the Fragile X-associated disorders.

Fragile X-associated disorders are Repeat Expansion Diseases that result from expansion of a CGG/CCG-repeat in the FMR1 gene. Contractions of the repeat tract also occur, albeit at lower frequency. However, these contractions can potentially modulate disease symptoms or generate an allele with repeat numbers in the normal range. Little is known about the expansion mechanism and even less about contractions. We have previously demonstrated that the mismatch repair (MMR) protein MSH2 is required for expansions in a mouse model of these disorders. Here, we show that MSH3, the MSH2-binding partner in the MutSß complex, is required for 98% of germ line expansions and all somatic expansions in this model. In addition, we provide evidence for two different contraction mechanisms that operate in the mouse model, a MutSß-independent one that generates small contractions and a MutSß-dependent one that generates larger ones. We also show that MutSß complexes formed with the repeats have altered kinetics of ATP hydrolysis relative to complexes with bona fide MMR substrates and that MutSß increases the stability of the CCG-hairpins at physiological temperatures. These data may have important implications for our understanding of the mechanism(s) of repeat instability and for the role of MMR proteins in this process.

Is thymidine glycol containing DNA a substrate of E. coli DNA mismatch repair system?

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

Oxidative stress and the DNA mismatch repair pathway.

SIGNIFICANCE: Living organisms are under constant assault by a combination of environmental and endogenous oxidative DNA damage, inducing the modification of proteins, lipids, and DNA. Failure to resolve these oxidative modifications is associated with genome instability and the development of many disease states. To maintain genomic integrity, oxidative lesions must be precisely targeted and efficiently resolved. For this, cells have evolved an intricate network of DNA repair mechanisms to detect and repair oxidative DNA damage. RECENT ADVANCES: Emerging evidence suggests that in addition to the base excision repair and nucleotide excision repair pathways, the DNA mismatch repair (MMR) pathway plays an important role in mediating oxidative DNA damage repair. Studies in lower organisms and mammalian cells have enabled us to further dissect this critical role and elucidate the precise mechanisms of repair. CRITICAL ISSUES: Identification of synthetic lethal interactions between MMR deficiency and the accumulation of oxidative DNA damage raises the tantalizing prospect that oxidative DNA-damaging agents may be utilized to selectively target MMR-deficient cancers and potentially other tumor types deficient for oxidative DNA repair molecules. FUTURE DIRECTIONS: In this review, we emphasize the clinical relevance and potential translation of exploiting this oxidative DNA repair mechanism using synthetic lethality studies in MMR-deficient cells, to develop improved treatment strategies that will benefit cancer patients.

ATR signalling: more than meeting at the fork.

Preservation of genome integrity via the DNA-damage response is critical to prevent disease. ATR (ataxia telangiectasia mutated- and Rad3-related) is essential for life and functions as a master regulator of the DNA-damage response, especially during DNA replication. ATR controls and co-ordinates DNA replication origin firing, replication fork stability, cell cycle checkpoints and DNA repair. Since its identification 15 years ago, a model of ATR activation and signalling has emerged that involves localization to sites of DNA damage and activation through protein-protein interactions. Recent research has added an increasingly detailed understanding of the canonical ATR pathway, and an appreciation that the canonical model does not fully capture the complexity of ATR regulation. In the present article, we review the ATR signalling process, focusing on mechanistic findings garnered from the identification of new ATR-interacting proteins and substrates. We discuss how to incorporate these new insights into a model of ATR regulation and point out the significant gaps in our understanding of this essential genome-maintenance pathway.

Epistatic roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in coping with reactive oxygen species-induced DNA damage.

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H(2)O(2))-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H(2)O(2)-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed.

Wider access to genotypic space facilitates loss of cooperation in a bacterial mutator.

Understanding the ecological, evolutionary and genetic factors that affect the expression of cooperative behaviours is a topic of wide biological significance. On a practical level, this field of research is useful because many pathogenic microbes rely on the cooperative production of public goods (such as nutrient scavenging molecules, toxins and biofilm matrix components) in order to exploit their hosts. Understanding the evolutionary dynamics of cooperation is particularly relevant when considering long-term, chronic infections where there is significant potential for intra-host evolution. The impact of responses to non-social selection pressures on social evolution is arguably an under-examined area. In this paper, we consider how the evolution of a non-social trait--hypermutability--affects the cooperative production of iron-scavenging siderophores by the opportunistic human pathogen Pseudomonas aeruginosa. We confirm an earlier prediction that hypermutability accelerates the breakdown of cooperation due to increased sampling of genotypic space, allowing mutator lineages to generate non-cooperative genotypes with the ability to persist at high frequency and dominate populations. This may represent a novel cost of hypermutability.

Mechanism of MutS searching for DNA mismatches and signaling repair.

DNA mismatch repair is initiated by the recognition of mismatches by MutS proteins. The mechanism by which MutS searches for and recognizes mismatches and subsequently signals repair remains poorly understood. We used single-molecule analyses of atomic force microscopy images of MutS-DNA complexes, coupled with biochemical assays, to determine the distributions of conformational states, the DNA binding affinities, and the ATPase activities of wild type and two mutants of MutS, with alanine substitutions in the conserved Phe-Xaa-Glu mismatch recognition motif. We find that on homoduplex DNA, the conserved Glu, but not the Phe, facilitates MutS-induced DNA bending, whereas at mismatches, both Phe and Glu promote the formation of an unbent conformation. The data reveal an unusual role for the Phe residue in that it promotes the unbending, not bending, of DNA at mismatch sites. In addition, formation of the specific unbent MutS-DNA conformation at mismatches appears to be required for the inhibition of ATP hydrolysis by MutS that signals initiation of repair. These results provide a structural explanation for the mechanism by which MutS searches for and recognizes mismatches and for the observed phenotypes of mutants with substitutions in the Phe-Xaa-Glu motif.

The role of MutS oligomers on Pseudomonas aeruginosa mismatch repair system activity.

The Escherichia coli DNA Mismatch Repair (MMR) protein MutS exist as dimers and tetramers in solution, and the identification of its functional oligomeric state has been matter of extensive study. In the present work, we have analyzed the oligomerization state of MutS from Pseudomonas aeruginosa a bacterial species devoid of Dam methylation and MutH homologue. By analyzing native MutS and different mutated versions of the protein, we determined that P. aeruginosa MutS is mainly tetrameric in solution and that its oligomerization capacity is conducted as in E. coli, by the C-terminal region of the protein. The analysis of mismatch oligonucleotide binding activity showed that wild-type MutS binds to DNA as tetramer. The DNA binding activity decreased when the C-terminal region was deleted (MutSDelta798) or when a full-length MutS with tetramerization defects (MutSR842E) was tested. The ATPase activity of MutSDelta798 was similar to MutSR842E and diminished respect to the wild-type protein. Experiments carried out on a P. aeruginosa mutS strain to test the proficiency of different oligomeric versions of MutS to function in vivo showed that MutSDelta798 is not functional and that full-length dimeric version MutSR842E, is not capable of completely restoring the MMR activity of the mutant strain. Additional experiments carried out in conditions of high mutation rate induced by the base analogue 2-AP confirm that the dimeric version of MutS is not as efficient as the tetrameric wild-type protein to prevent mutations. Therefore, it is concluded that although dimeric MutS is sufficient for MMR activity, optimal activity is obtained with the tetrameric version of the protein and therefore it should be considered as the active form of MutS in P. aeruginosa.

Role of hypermutability in the evolution of the genus Oenococcus.

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium primarily responsible for malolactic fermentation in wine. A recent comparative genomic analysis of O. oeni PSU-1 with other sequenced lactic acid bacteria indicates that PSU-1 lacks the mismatch repair (MMR) genes mutS and mutL. Consistent with the lack of MMR, mutation rates for O. oeni PSU-1 and a second oenococcal species, O. kitaharae, were higher than those observed for neighboring taxa, Pediococcus pentosaceus and Leuconostoc mesenteroides. Sequence analysis of the rpoB mutations in rifampin-resistant strains from both oenococcal species revealed a high percentage of transition mutations, a result indicative of the lack of MMR. An analysis of common alleles in the two sequenced O. oeni strains, PSU-1 and BAA-1163, also revealed a significantly higher level of transition substitutions than were observed in other Lactobacillales species. These results suggest that the genus Oenococcus is hypermutable due to the loss of mutS and mutL, which occurred with the divergence away from the neighboring Leuconostoc branch. The hypermutable status of the genus Oenococcus explains the observed high level of allelic polymorphism among known O. oeni isolates and likely contributed to the unique adaptation of this genus to acidic and alcoholic environments.

Mismatch repair proteins as sensors of alkylation DNA damage.

The DNA mismatch repair (MMR) system maintains genome integrity by correcting replication errors. MMR also stimulates checkpoint and cell death responses to DNA damage suggested by the resistance of MMR-defective tumor cells to several chemotherapeutic agents. MMR-dependent cytotoxic response may result from futile repair; however, MMR-mediated apoptosis has been genetically separated from its repair function. In a recent issue of Molecular Cell, Yoshioka and coworkers show that MMR complexes (MutSalpha and MutLalpha) are required for the recruitment of ATR-ATRIP to sites of alkylation damage, demonstrating that MMR complexes can function as sensors in DNA damage signal transduction.

DNA mismatch repair system. Classical and fresh roles.

The molecular mechanisms of the DNA mismatch repair (MMR) system have been uncovered over the last decade, especially in prokaryotes. The results obtained for prokaryotic MMR proteins have provided a framework for the study of the MMR system in eukaryotic organisms, such as yeast, mouse and human, because the functions of MMR proteins have been conserved during evolution from bacteria to humans. However, mutations in eukaryotic MMR genes result in pleiotropic phenotypes in addition to MMR defects, suggesting that eukaryotic MMR proteins have evolved to gain more diverse and specific roles in multicellular organisms. Here, we summarize recent advances in the understanding of both prokaryotic and eukaryotic MMR systems and describe various new functions of MMR proteins that have been intensively researched during the last few years, including DNA damage surveillance and diversification of antibodies.

Sequence variability and functional analysis of MutS of hypermutable Pseudomonas aeruginosa cystic fibrosis isolates.

In this study, we investigated the variability of MutS among Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patients. Sequencing of the mutS gene of 15 hypermutable P. aeruginosa isolates obtained from different patients revealed high rates of nucleotide substitutions as compared to that of strain PAO1. Significantly more synonymous than non-synonymous nucleotide substitutions have been found, indicating that generally MutS is highly conserved. The functional analysis of MutS variants by complementation of a PAO1 mutS mutant revealed 5 isolates with a defective MutS due to frameshift mutations or amino acid substitutions. This work supports the hypothesis that the respiratory tract of CF patients represents an environment that favors the selection of highly adaptive mutator phenotypes.

The Helicobacter pylori MutS protein confers protection from oxidative DNA damage.

The human gastric pathogenic bacterium Helicobacter pylori lacks a MutSLH-like DNA mismatch repair system. Here, we have investigated the functional roles of a mutS homologue found in H. pylori, and show that it plays an important physiological role in repairing oxidative DNA damage. H. pylori mutS mutants are more sensitive than wild-type cells to oxidative stress induced by agents such as H2O2, paraquat or oxygen. Exposure of mutS cells to oxidative stress results in a significant ( approximately 10-fold) elevation of mutagenesis. Strikingly, most mutations in mutS cells under oxidative stress condition are G:C to T:A transversions, a signature of 8-oxoguanine (8-oxoG). Purified H. pylori MutS protein binds with a high specific affinity to double-stranded DNA (dsDNA) containing 8-oxoG as well as to DNA Holliday junction structures, but only weakly to dsDNA containing a G:A mismatch. Under oxidative stress conditions, mutS cells accumulate higher levels (approximately threefold) of 8-oxoG DNA lesions than wild-type cells. Finally, we observe that mutS mutant cells have reduced colonization capacity in comparison to wild-type cells in a mouse infection model.

Functional characterization of the DNA mismatch binding protein MutS from Haemophilus influenzae.

This investigation demonstrates DNA mismatch repair activity in Haemophilus influenzae cell free extracts. The mutS gene as well as purified protein of H. influenzae restored repair activity in complementation assays performed with mutS deficient Escherichia coli strain. The difference in affinity for GT and AC mismatched bases by H. influenzae MutS was reflected in the efficiency with which these DNA heteroduplexes were repaired in vitro, with GT being repaired well and AC the least. Unlike E. coli MutS, the H. influenzae homolog failed to give protein-DNA complex with homoduplex DNA. Interestingly, MutS was found to bind single-stranded DNA but with lesser affinity as compared to heteroduplex DNA. Apart from the nucleotide- and DNA-mediated conformational transitions, as monitored by circular dichroism and limited proteolysis, our data suggest a functional role when H. influenzae MutS encounters single-stranded DNA during exonucleolytic step of DNA repair process. We propose that, conformational changes in H. influenzae MutS not only modulate mismatch recognition but also trigger some of the down stream processes involved in the DNA mismatch repair process.