Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2-Msh6 mismatch repair protein

Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2-Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S1-but which of these subunits, S1 or S2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2-Msh6 heterodimer (in Msh2-Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a "cis" linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2-Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch.     

(CAG)(n)-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition

Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-Msh3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-Msh3-CAG hairpin complex that could misdirect the DNA repair process.     

DNA binding properties of the yeast Msh2-Msh6 and Mlh1-Pms1 heterodimers

We describe here our recent studies of the DNA binding properties of Msh2-Msh6 and Mlh1-Pms1, two protein complexes required to repair mismatches generated during DNA replication. Mismatched DNA binding by Msh2-Msh6 was probed by mutagenesis based on the crystal structure of the homologous bacterial MutS homodimer bound to DNA. The results suggest that several amino acid side chains inferred to interact with the DNA backbone near the mismatch are critical for repair activity. These contacts, which are different in Msh2 and Msh6, likely facilitate stacking and hydrogen bonding interactions between side chains in Msh6 and the mismatched base, thus stabilizing a kinked DNA conformation that permits subsequent repair steps coordinated by the Mlh1-Pms1 heterodimer. Mlh1-Pms1 also binds to DNA, but independently of a mismatch. Mlh1-Pms1 binds short DNA substrates with low affinity and with a slight preference for single-stranded DNA. It also binds longer duplex DNA molecules, but with a higher affinity indicative of cooperative binding. Indeed, imaging by atomic force microscopy reveals cooperative DNA binding and simultaneous interaction with two DNA duplexes. The novel DNA binding properties of Mlh1-Pms1 may be relevant to signal transduction during DNA mismatch repair and to recombination, meiosis and cellular responses to DNA damage.     

Msh2-Msh3 interferes with Okazaki fragment processing to promote trinucleotide repeat expansions

Trinucleotide repeat (TNR) expansions are the underlying cause of more than 40 neurodegenerative and neuromuscular diseases, including myotonic dystrophy and Huntington's disease. Although genetic evidence points to errors in DNA replication and/or repair as the cause of these diseases, clear molecular mechanisms have not been described. Here, we 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. Furthermore, we 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.     

Dynamic basis for one-dimensional DNA scanning by the mismatch repair complex Msh2-Msh6

The ability of proteins to locate specific sites or structures among a vast excess of nonspecific DNA is a fundamental theme in biology. Yet the basic principles that govern these mechanisms remain poorly understood. For example, mismatch repair proteins must scan millions of base pairs to find rare biosynthetic errors, and they then must probe the surrounding region to identify the strand discrimination signals necessary to distinguish the parental and daughter strands. To determine how these proteins might function we used single-molecule optical microscopy to answer the following question: how does the mismatch repair complex Msh2-Msh6 interrogate undamaged DNA? Here we show that Msh2-Msh6 slides along DNA via one-dimensional diffusion. These findings indicate that interactions between Msh2-Msh6 and DNA are dominated by lateral movement of the protein along the helical axis and have implications for how MutS family members travel along DNA at different stages of the repair reaction.     

The ubiquitously expressed DNA-binding protein late SV40 factor binds Ig switch regions and represses class switching to IgA

Ig heavy chain class switch recombination (CSR) determines the expression of Ig isotypes. The molecular mechanism of CSR and the factors regulating this process have remained elusive. Recombination occurs primarily within switch (S) regions, located upstream of each heavy chain gene (except Cdelta). These repetitive sequences contain consensus DNA-binding sites for the DNA-binding protein late SV40 factor (LSF) (CP2/leader-binding protein-1c). In this study, we demonstrate by EMSA that purified rLSF, as well as LSF within B cell extracts, directly binds both Smu and Salpha sequences. To determine whether LSF is involved in regulating CSR, two different LSF dominant negative variants were stably expressed in the mouse B cell line I.29 mu, which can be induced to switch from IgM to IgA. Overexpression of these dominant negative LSF proteins results in decreased levels of endogenous LSF DNA-binding activity and an increase in cells undergoing CSR. Thus, LSF represses class switching to IgA. In agreement, LSF DNA-binding activity was found to decrease in whole cell extracts from splenic B cells induced to undergo class switching. To elucidate the mechanism of CSR regulation by LSF, the interactions of LSF with proteins involved in chromatin modification were tested in vitro. LSF interacts with both histone deacetylases and the corepressor Sin3A. We propose that LSF represses CSR by histone deacetylation of chromatin within S regions, thereby limiting accessibility to the switch recombination machinery.     

Mismatch recognition-coupled stabilization of Msh2-Msh6 in an ATP-bound state at the initiation of DNA repair

Mismatch repair proteins correct errors in DNA via an ATP-driven process. In eukaryotes, the Msh2-Msh6 complex recognizes base pair mismatches and small insertion/deletions in DNA and initiates repair. Both Msh2 and Msh6 proteins contain Walker ATP-binding motifs that are necessary for repair activity. To understand how these proteins couple ATP binding and hydrolysis to DNA binding/mismatch recognition, the ATPase activity of Saccharomyces cerevisiae Msh2-Msh6 was examined under pre-steady-state conditions. Acid-quench experiments revealed that in the absence of DNA, Msh2-Msh6 hydrolyzes ATP rapidly (burst rate = 3 s(-1) at 20 degrees C) and then undergoes a slow step in the pathway that limits catalytic turnover (k(cat) = 0.1 s(-1)). ATP is hydrolyzed similarly in the presence of fully matched duplex DNA; however, in the presence of a G:T mismatch or +T insertion-containing DNA, ATP hydrolysis is severely suppressed (rate = 0.1 s(-1)). Pulse-chase experiments revealed that Msh2-Msh6 binds ATP rapidly in the absence or in the presence of DNA (rate = 0.1 microM(-1) s(-1)), indicating that for the Msh2-Msh6.mismatched DNA complex, a step after ATP binding but before or at ATP hydrolysis is the rate-limiting step in the pathway. Thus, mismatch recognition is coupled to a dramatic increase in the residence time of ATP on Msh2-Msh6. This mismatch-induced, stable ATP-bound state of Msh2-Msh6 likely signals downstream events in the repair pathway.     

The Saccharomyces cerevisiae Msh2 and Msh6 proteins form a complex that specifically binds to duplex oligonucleotides containing mismatched DNA base pairs.

The yeast Saccharomyces cerevisiae encodes six proteins, Msh1p to Msh6p, that show strong amino acid sequence similarity to MutS, a central component of the bacterial mutHLS mismatch repair system. Recent studies with humans and S. cerevisiae suggest that in eukaryotes, specific MutS homolog complexes that display unique DNA mismatch specificities exist. In this study, the S. cerevisiae 109-kDa Msh2 and 140-kDa Msh6 proteins were cooverexpressed in S. cerevisiae and shown to interact in an immunoprecipitation assay and by conventional chromatography. Deletion analysis of MSH2 indicated that the carboxy-terminal 114 amino acids of Msh2p are important for Msh6p interaction. Purified Msh2p-Msh6p selectively bound to duplex oligonucleotide substrates containing a G/T mismatch and a +1 insertion mismatch but did not show specific binding to +2 and +4 insertion mismatches. The mismatch binding specificity of the Msh2p-Msh6p complex, as measured by on-rate and off-rate binding studies, was abolished by ATP. Interestingly, palindromic substrates that are poorly repaired in vivo were specifically recognized by Msh2p-Msh6p; however, the binding of Msh2p-Msh6p to these substrates was not modulated by ATP. Taken together, these studies suggest that the repair of a base pair mismatch by the Msh2p-Msh6p complex is dependent on the ability of the Msh2p-Msh6p-DNA mismatch complex to use ATP hydrolysis to activate downstream events in mismatch repair.     

Engineered Disulfide-forming Amino Acid Substitutions Interfere with a Conformational Change in the Mismatch Recognition Complex Msh2-Msh6 Required for Mismatch Repair

ATP binding causes the mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces conformational changes in Msh2-Msh6; however, the nature of these conformational changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a conformational change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue Arg¹¹²⁴. An engineered disulfide bond within this region prevented the ATP-driven conformational change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven conformational change plays a role in mismatch repair.     

Dominant Saccharomyces cerevisiae msh6 mutations cause increased mispair binding and decreased dissociation from mispairs by Msh2-Msh6 in the presence of ATP

A previous study described four dominant msh6 mutations that interfere with both the Msh2-Msh6 and Msh2-Msh3 mismatch recognition complexes (Das Gupta, R., and Kolodner, R. D. (2000) Nat. Genet. 24, 53-56). Modeling predicted that two of the amino acid substitutions (G1067D and G1142D) interfere with protein-protein interactions at the ATP-binding site-associated dimer interface, one (S1036P) similarly interferes with protein-protein interactions and affects the Msh2 ATP-binding site, and one (H1096A) affects the Msh6 ATP-binding site. The ATPase activity of the Msh2-Msh6-G1067D and Msh2-Msh6-G1142D complexes was inhibited by GT, +A, and +AT mispairs, and these complexes showed increased binding to GT and +A mispairs in the presence of ATP. The ATPase activity of the Msh2-Msh6-S1036P complex was inhibited by a GT mispair, and it bound the GT mispair in the presence of ATP, whereas its interaction with insertion mispairs was unchanged compared with the wild-type complex. The ATPase activity of the Msh2-Msh6-H1096A complex was generally attenuated, and its mispair-binding behavior was unaffected. These results are in contrast to those obtained with the wild-type Msh2-Msh6 complex, which showed mispair-stimulated ATPase activity and ATP inhibition of mispair binding. These results indicate that the dominant msh6 mutations cause more stable binding to mispairs and suggest that there may be differences in how base base and insertion mispairs are recognized.     

Mutations occur in the Ig Smu region but rarely in Sgamma regions prior to class switch recombination

Nucleotide substitutions are found in recombined Ig switch (S) regions and also in unrecombined (germline, GL) Smicro segments in activated splenic B cells. Herein we examine whether mutations are also introduced into the downstream acceptor S regions prior to switch recombination, but find very few mutations in GL Sgamma3 and Sgamma1 regions in activated B cells. These data suggest that switch recombination initiates in the Smicro segment and secondarily involves the downstream acceptor S region. Furthermore, the pattern and specificity of mutations in GL and recombined Smicro segments differ, suggesting different repair mechanisms. Mutations in recombined Smicro regions show a strong bias toward G/C base pairs and WRCY/RGYW hotspots, whereas mutations introduced into the GL Smicro do not. Additionally, induction conditions affect mutation specificity within the GL Smicro segment. Mutations are most frequent near the S-S junctions and decrease rapidly with distance from the junction. Finally, we find that mice expressing a transgene for terminal deoxynucleotidyl transferase (TdT) have nucleotide insertions at S-S junctions, indicating that the recombining DNA ends are accessible to end-processing enzyme activities.     

Syntaxin 1A binds to the cytoplasmic C terminus of Kv2.1 to regulate channel gating and trafficking

Voltage-gated K(+) (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet beta-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet beta-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet beta-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634-853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca(2+) channels. In contrast to Ca(2+) channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.     

The Drosophila zeste protein binds cooperatively to sites in many gene regulatory regions: implications for transvection and gene regulation.

The Drosophila zeste protein binds in vitro to several sites in the white, Ultrabithorax, decapentaplegic, Antennapedia, and engrailed genes and to at least one site in the zeste gene itself. The distribution of these sites corresponds often with that of regulatory elements in these genes as defined by mutations or, in the case of white, by molecular analysis. A zeste binding site is frequently found in the immediate vicinity of the promoter. zeste binding sites are composed of two or more zeste recognition sequences T/CGAGT/CG. Isolated consensus sequences do not bind or footprint. Cooperative interactions are involved both in binding to a given site and between proteins bound at independent sites. zeste bound to one DNA molecule can in fact bind simultaneously to another DNA molecule. These results suggest a general role for zeste in bringing together distant regulatory elements controlling the activity of a target gene. In this model, transvection effects are a by-product of normal intragenic zeste action.     

A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels

Store-operated Ca²⁺ entry (SOCE) is a universal mechanism to increase intracellular Ca²⁺ concentrations in non-excitable cells. It is initiated by the depletion of ER Ca²⁺ stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca²⁺ release activated Ca²⁺ (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca²⁺ influx and CRAC currents (I_CRAC) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I_CRAC consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca²⁺ stores.     

The carboxyl terminus of the cystic fibrosis transmembrane conductance regulator binds to AP-2 clathrin adaptors

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes rapid and efficient endocytosis. Since functionally active CFTR is found in purified clathrin-coated vesicles isolated from both cultured epithelial cells and intact epithelial tissues, we investigated the molecular mechanisms whereby CFTR could enter such endocytic clathrin-coated vesicles. In vivo cross-linking and in vitro pull-down assays show that full-length CFTR binds to the endocytic adaptor complex AP-2. Fusion proteins containing the carboxyl terminus of CFTR (amino acids 1404-1480) were also able to bind AP-2 but did not bind the Golgi-specific adaptor complex AP-1. Substitution of an alanine residue for tyrosine at position 1424 significantly reduced the ability of AP-2 to bind the carboxyl terminus of CFTR; however, mutation to a phenylalanine residue (an amino acid found at position 1424 in dogfish CFTR) did not perturb AP-2 binding. Secondary structure predictions suggest that Tyr(1424) is present in a beta-turn conformation, a conformation disrupted by alanine but not phenylalanine. Together, these data suggest that the carboxyl terminus of CFTR contains a tyrosine-based internalization signal that interacts with the endocytic adaptor complex AP-2 to facilitate efficient entry of CFTR into clathrin-coated vesicles.