Summary Trinucleotide repeat (TNR) expansions are the underlying cause of more than forty neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington’s disease. Although genetic evidence has attributed the cause of these diseases to errors in DNA replication and/or repair, clear molecular mechanisms have not been described. We have focused on the role of the mismatch repair complex Msh2-Msh3 in promoting TNR expansions. We demonstrate that Msh2-Msh3 promotes CTG and CAG repeat expansions in vivo in Saccharomyces cerevisiae. We further provide biochemical evidence that Msh2-Msh3 directly interferes with normal Okazaki fragment processing by flap endonuclease1 (Rad27) and DNA Ligase I (Cdc9) in the presence of TNR sequences, thereby producing small, incremental expansion events. We believe that this is the first mechanistic evidence showing the interplay of replication and repair proteins in the expansion of sequences during lagging strand DNA replication.

DNA-binding proteins search for specific targets via facilitated diffusion along a crowded genome. However, little is known about how crowded DNA modulates facilitated diffusion and target recognition. Here we use DNA curtains and single-molecule fluorescence imaging to investigate how Msh2–Msh3, a eukaryotic mismatch repair complex, navigates on crowded DNA. Msh2–Msh3 hops over nucleosomes and other protein roadblocks, but maintains sufficient contact with DNA to recognize a single lesion. In contrast, Msh2–Msh6 slides without hopping and is largely blocked by protein roadblocks. Remarkably, the Msh3-specific mispair-binding domain (MBD) licences a chimeric Msh2–Msh6(3MBD) to bypass nucleosomes. Our studies contrast how Msh2–Msh3 and Msh2–Msh6 navigate on a crowded genome and suggest how Msh2–Msh3 locates DNA lesions outside of replication-coupled repair. These results also provide insights into how DNA repair factors search for DNA lesions in the context of chromatin.

In Saccharomyces cerevisiae, repair of insertion/deletion loops is carried out by Msh2-Msh3-mediated mismatch repair (MMR). Msh2-Msh3 is also required for 3’ non-homologous tail removal (3’NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, the kinetics of the two processes appear different; MMR is likely rapid in order to coordinate with the replication fork, whereas 3’ NHTR has been shown to be a slower process. To understand the molecular requirements in both repair pathways, we performed an in vivo analysis of well conserved residues in Msh3 that are hypothesized to be required for MMR and/or 3’NHTR. These residues are predicted to be involved in either communication between the DNA-binding and ATPase domains within the complex or nucleotide binding and/or exchange within Msh2-Msh3. We identified a set of aromatic residues within the FLY motif of the predicted Msh3 nucleotide binding pocket that are essential for Msh2-Msh3-mediated MMR but are largely dispensable for 3’NHTR. In contrast, mutations in other regions gave similar phenotypes in both assays. Based on these results, we suggest the two pathways have distinct requirements with respect to the position of the bound ATP within Msh3. We propose that the differences are related, at least in part, to the kinetics of each pathway. Proper binding and positioning of ATP is required to induce rapid conformational changes at the replication fork, but is less important when more time is available for repair, as in 3’ NHTR.

WILL[AMS. Can. J. Chem. 62, 755 (1984). The ruthenium(l1) porphyrin complex R U ( O E P ) ( P P~~)~ (OEP = the dianion of octaethylporphyrin) has been prepared from Ru(OEP)(CO)EtOH, and the X-ray crystal structure determined; as expected, the six-coordinate ruthenium is situated in the porphyrin plane and has two axial phosphine ligands. Synthesized also from the carbonyl(ethano1) precursors were the corresponding tris(p-methoxypheny1)phosphine complex. and the Ru(TPP)L, (TPP = the dianion of tetraphenylporphyrin, L = PPh,, P(p-CH30C6H,),, PnBu3) and Ru(TPP)(CO)PPh3 complexes. Optical and 'H nmr data are presented for the complexes in solution. In some cases dissociation of a phosphine ligand to generate five-coordinate species occurs and this has been studied quantitatively in toluene at 20°C for the Ru(OEP)L2 and Ru(TPP)L2 systems. ~ Introduction Our continuing interest in, and development of, ruthenium porphyrin chemistry (1) led us to discover that ruthenium(I1) porphyrins containing tertiary phosphines as axial ligands were efficient catalysts for the decarbonylation of aldehydes (2), as well as for oxidation of substrates such as phosphines and sulfides by molecular oxygen through generation of hydrogen peroxide (3). The complexes also undergo two successive oneelectron electrochemical oxidations to yield first a ruthenium(11I) bisphosphine cation and then a ruthenium(II1) r-cation radical (4).We report here the details of the preparation and characterization of some of the ruthenium porphyrin -tertiary phosphine complexes, including the X-ray structure of RU(OEP)(PP~~),.? The dissociative equilibrium involving the loss of a phosphine ligand from the Ru(porp)L,, L = PPh,, P(p-CH30C6H4)3, complexes in toluene solution is also considered. Other groups ( 5 , 6) have described the synthesis of Ru-(TPP)(PPh,),, but the reported solution optical spectral data did not make allowance for the dissociation of phosphine.

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Bis($-cyclopentadienyl)zirconium(IV) alkyl chlorides and hydrides have been prepared and characterized. Hydrogenation of these species yields the corresponding alkane and zirconium hydride complexes, Cp,ZrHCl and Cp2ZrH2, respectively. Deuterium labeling experiments suggest that these do complexes activate H, by heterolytic attack on that molecule.Qualitative rates for hydrogenation of a series of complexes were Cp,Zr(R)H > Cp,Zr(R)Cl N Cp,ZrR, > [(Cp2ZrC1)2-(p-OCHR)] > Cp,Zr(COR)Cl. This rate trend is the same as that for carbonylation and suggests a conceptual link between mechanisms for hydrogenation and carbonylation of these unsaturated complexes. A possible relationship is noted between heterolytic activation and oxidative addition of H2 to transition-metal species.

In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′ NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′ NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3′ NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.

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