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The MutS gene family is distributed across the tree of life and is involved in recombination, DNA repair, and protein translation. Multiple evolutionary processes have expanded the set of MutS genes in plants relative to other eukaryotes. Here, we investigate the origins and functions of these plant-specific genes. Land plants, green algae, red algae, and glaucophytes share cyanobacterial-like MutS1 and MutS2 genes that presumably were gained via plastid endosymbiotic gene transfer. MutS1 was subsequently lost in some taxa, including seed plants, whereas MutS2 was duplicated in Viridiplantae (i.e., land plants and green algae) with widespread retention of both resulting paralogs. Viridiplantae also have two anciently duplicated copies of the eukaryotic MSH6 gene (i.e., MSH6 and MSH7 ) and acquired MSH1 via horizontal gene transfer - potentially from a nucleocytovirus. Despite sharing the same name, "plant MSH1 " is not directly related to the gene known as MSH1 in some fungi and animals, which may be an ancestral eukaryotic gene acquired via mitochondrial endosymbiosis and subsequently lost in most eukaryotic lineages. There has been substantial progress in understanding the functions of MSH1 and MSH6 / MSH7 in plants, but the roles of the cyanobacterial-like MutS1 and MutS2 genes remain uncharacterized. Known functions of bacterial homologs and predicted protein structures, including fusions to diverse nuclease domains, provide hypotheses about potential molecular mechanisms. Because most plant-specific MutS proteins are targeted to the mitochondria and/or plastids, the expansion of this family appears to have played a large role in shaping plant organelle genetics.

During meiosis, induction of DNA double strand breaks (DSB) leads to recombination between homologous chromosomes, resulting in crossovers (CO) and non-crossovers (NCO). In the mouse, only 10% of DSBs resolve as COs, mostly through a class I pathway dependent on MutSγ (MSH4/ MSH5) and MutLγ (MLH1/MLH3), the latter representing the ultimate marker of these CO events. A second Class II CO pathway accounts for only a few COs, but is not thought to involve MutSγ/ MutLγ, and is instead dependent on MUS81-EME1. For class I events, loading of MutLγ is thought to be dependent on MutSγ, however MutSγ loads very early in prophase I at a frequency that far exceeds the final number of class I COs. Moreover, loss of MutSγ in mouse results in apoptosis before CO formation, preventing the analysis of its CO function. We generated a mutation in the ATP binding domain of Msh5 (Msh5GA). While this mutation was not expected to affect MutSγ complex formation, MutSγ foci do not accumulate during prophase I. However, most spermatocytes from Msh5GA/GA mice progress to late pachynema and beyond, considerably further than meiosis in Msh5−/− animals. At pachynema, Msh5GA/GA spermatocytes show persistent DSBs, incomplete homolog pairing, and fail to accumulate MutLγ. Unexpectedly, Msh5GA/GA diakinesis-staged spermatocytes have no chiasmata at all from any CO pathway, indicating that a functional MutSγ complex is critical for all CO events regardless of their mechanism of generation.

During meiosis, induction of DNA double strand breaks (DSB) leads to recombination between homologous chromosomes, resulting in crossovers (CO) and non-crossovers (NCO). In the mouse, only 10% of DSBs resolve as COs, mostly through a class I pathway dependent on MutSg (MSH4/MSH5) and MutLg (MLH1/MLH3), the latter representing the ultimate marker of these CO events. A second Class II CO pathway accounts for only a few COs, but is not thought to involve MutSg/MutLg, and is instead dependent on MUS81-EME1. For class I events, loading of MutLg is thought to be dependent on MutSg, however MutSg loads very early in prophase I at a frequency that far exceeds the final number of class I COs. Moreover, loss of MutSg in mouse results in apoptosis before CO formation, preventing the analysis of its CO function. We generated a mutation in the ATP binding domain of Msh5 (Msh5GA). While this mutation was not expected to affect MutSg complex formation, MutSg foci do not accumulate during prophase I. However, most spermatocytes from Msh5GA/GA mice progress to late pachynema and beyond, considerably further than meiosis in Msh52/2 animals. At pachynema, Msh5GA/GA spermatocytes show persistent DSBs, incomplete homolog pairing, and fail to accumulate MutLg. Unexpectedly, Msh5GA/GA diakinesis-staged spermatocytes have no chiasmata at all from any CO pathway, indicating that a functional MutSg complex is critical for all CO events regardless of their mechanism of generation. [ABSTRACT FROM AUTHOR]

Abstract: DNA mismatch repair (MMR) of simple base mismatches and small insertion-deletion loops in eukaryotes is initiated by the binding of the MutS homolog 2 (MSH2)-MSH6 heterodimer to mismatched DNA. Cadmium (Cd) is a genotoxic heavy metal that has been recognized as a human carcinogen. Oxidant stress and inhibition of DNA repair have been proposed as major factors underlying Cd genotoxicity. Our previous studies indicated the ability of Cd to disturb the gene expression of MSH6 in zebrafish (Danio rerio) embryos. This study was undertaken to explore if Cd-induced oxidative stress down-regulated MSH gene activities. Following the exposure of zebrafish embryos at 1h post fertilization (hpf) to sublethal concentrations of Cd at 3–5μM for 4 or 9h, a parallel down-regulation of MSH2, MSH6 and Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene expression was detected by real-time RT-PCR and the expression levels were 40–50% of control after a 9-h exposure. Cd exposure also induced oxidative stress, yet no inhibition of catalase gene activity was observed. Whole mount in situ hybridization revealed a wide distribution of msh6 mRNA in the head regions of 10 hpf embryos and pretreatment of embryos with antioxidants butylhydroxytoluene (BHT), d-mannitol or N-acetylcysteine (NAC) at 1–10μM restored Cd-suppressed msh6 expression. QPCR confirmed the protective effects of antioxidants on Cd-suppressed msh2/msh6 mRNA production. Down-regulated MSH gene activities reaching about 50% of control were also induced in embryos exposed to paraquat, a reactive oxygen species (ROS)-generating herbicide, or hydrogen peroxide at 200μM. Hence, Cd at sublethal levels down-regulates msh2/msh6 expression primarily via ROS as signaling molecules. The transcriptional activation of human msh6 is known to be fully dependent on the specificity factor 1 (Sp1). Cd failed to inhibit the DNA binding activity of zebrafish Sp1 unless at lethal concentrations based on band shift assay, therefore excluding the involvement of Sp1 inactivation in Cd-induced MSH gene inhibition in zebrafish embryos. [Copyright &y& Elsevier]

Abstract: Avoidance of apoptosis is one of the hallmarks of cancer development and progression. Chemotherapeutic agents aim to initiate an apoptotic response, but often fail due to dysregulation. MSH proteins are capable of recognizing cisplatin damage in DNA and participate in the initiation of cell death. We have exploited this recognition and computationally simulated a MutS homolog (MSH) “death conformation”. Screening and docking experiments based on this model determined that the MSH2-dependent cell-death pathway can be induced by a small molecule without DNA damage, reserpine. Reserpine was identified via virtual screening on structures obtained from molecular dynamics as a small molecule that selectively binds a protein “death” conformation. The virtual screening predicts that this small molecule binds in the absence of DNA. Cell biology confirmed that reserpine triggers the MSH2-dependent cell-death pathway. This result supports the hypothesis that the MSH2-dependent pathway is initiated by specific protein conformational changes triggered by binding to either DNA damage or small compound molecules. These findings have multiple implications for drug discovery and cell biology. Computational modeling may be used to identify and eventually design small molecules that selectively activate particular pathways through conformational control. Molecular dynamics simulations can be used to model the biologically relevant conformations and virtual screening can then be used to select for small molecules that bind specific conformations. The ability of a small molecule to induce the cell-death pathway suggests a broader role for MMR proteins in cellular events, such as cell-death pathways, than previously suspected. [Copyright &y& Elsevier]

Stability of the mitochondrial genome is controlled by nuclear loci. In plants, nuclear genes suppress mitochondrial DNA rearrangements during development. One nuclear gene involved in this process is Msh1. Msh1 appears to be involved in the suppression of illegitimate recombination in plant mitochondria. To test the hypothesis that Msh1 disruption leads to the type of mitochondrial DNA rearrangements associated with naturally occurring cytoplasmic male sterility in plants, a transgenic approach for RNAi was used to modulate expression of Msh1 in tobacco and tomato. In both species, these experiments resulted in reproducible mitochondrial DNA rearrangements and a condition of male (pollen) sterility. The male sterility was, in each case, heritable, associated with normal female fertility, and apparently maternal in its inheritance. Segregation of the transgene did not reverse the male sterile phenotype, producing stable, non-transgenic male sterility. The reproducible transgenic induction of mitochondrial rearrangements in plants is unprecedented, providing a means to develop novel cytoplasmic male sterile lines for release as non-GMO or transgenic materials. [ABSTRACT FROM AUTHOR]

Abstract: MSH5 is known to play functional roles in an array of cellular processes such as DNA damage response and meiotic homologous recombination. Here, we report the characterization of an hMSH5 splicing variant (hMSH5sv) that resulted from the retention of the last 51bp of hMSH5 intron 6, in which the encoded 17-amino acid insertion between codons 179 and 180 does not compromise its capability to interact with hMSH4. We have also identified an hMSH5 polymorphism (C84T) that altered codon 29 of the hMSH5 gene resulting in a proline-to-serine change (P29S). The interaction domains of hMSH4 and hMSH5 have also been resolved. The P29S alteration is located within the interacting domain and leads to a weakened protein interaction with hMSH4. Together, our present study revealed the existence of two forms of hMSH5 variants in human cells. The different properties associated with these two hMSH5 variants underscore the potential functional diversity of the human hMSH5 gene. [Copyright &y& Elsevier]

Eukaryotic mismatch repair of simple base mispairs and small insertion-deletion loops is activated by the binding of a heterodimeric complex composed of MutS homolog 2(MSH2) and MSH6. Here we report the cloning of zebrafish (Danio rerio) MSH2 (zMSH2) cDNA that has an open reading frame of 2811 nucleotides encoding a polypeptide of 936 amino acids. The deduced amino acid sequence of zMSH2 shares a 69% identity to both human and mouse MSH2. The zMSH2 protein contains a putative tyrosine-42 mismatch-contacting residue located at the N-terminal mismatch recognition region and four C-terminal ATP-binding consensus sequences conserved among MutS homologs. The 105-kDa recombinant zMSH2 bound apparently stronger to a G–T heteroduplex than to a homoduplex probe as shown by a gel shift assay. A preferential expression of both zMSH2 and zMSH6 mRNA in early embryos was found by Northern blot analysis. Whole mount in situ hybridization revealed a major expression of zMSH2 in different regions of the brain, including eyes, telencephalon, and the fourth ventricle in 12- to 48-h-old embryos. The production of zMSH2 mRNA gradually decreased in more mature 60- to 120-h-old zebrafish, reflecting a positive correlation between the amount of proliferating cells and MSH gene expression. [Copyright &y& Elsevier]