Browse
Submit Data
Databases
API
Help

Dataset Information

25 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The N terminus of Saccharomyces cerevisiae Msh6 is an unstructured tether to PCNA.

ABSTRACT: The eukaryotic MutS homolog complexes, Msh2-Msh6 and Msh2-Msh3, recognize mismatched bases in DNA during mismatch repair (MMR). The eukaryote-specific N-terminal regions (NTRs) of Msh6 and Msh3 have not been characterized other than by demonstrating that they contain an N-terminal PCNA-interacting motif. Here we have demonstrated genetically that the NTR of Msh6 has an important role in MMR that is partially redundant with PCNA binding. Small-angle X-ray scattering (SAXS) was used to determine the solution structure of the complex of PCNA with Msh2-Msh6 and with the isolated Msh6 NTR, revealing that the Msh6 NTR is a natively disordered domain that forms an extended tether between Msh6 and PCNA. Moreover, computational analysis of PCNA-interacting motifs in the S. cerevisiae proteome indicated that flexible linkers are a common theme for PCNA-interacting proteins that may serve to localize these binding partners without tightly restraining them to the immediate vicinity of PCNA.

SUBMITTER: Shell SS

PROVIDER: S-EPMC2001284 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

The N terminus of Saccharomyces cerevisiae Msh6 is an unstructured tether to PCNA.

Shell Scarlet S SS Putnam Christopher D CD Kolodner Richard D RD

Molecular cell 20070501 4

The eukaryotic MutS homolog complexes, Msh2-Msh6 and Msh2-Msh3, recognize mismatched bases in DNA during mismatch repair (MMR). The eukaryote-specific N-terminal regions (NTRs) of Msh6 and Msh3 have not been characterized other than by demonstrating that they contain an N-terminal PCNA-interacting motif. Here we have demonstrated genetically that the NTR of Msh6 has an important role in MMR that is partially redundant with PCNA binding. Small-angle X-ray scattering (SAXS) was used to determine t ...[more]

PMID: 17531814

Similar Datasets

Interaction between the Msh2 and Msh6 nucleotide-binding sites in the Saccharomyces cerevisiae Msh2-Msh6 complex.

Project description: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.

| S-EPMC2838348 | biostudies-literature

Reversal of PCNA ubiquitylation by Ubp10 in Saccharomyces cerevisiae.

Project description:Regulation of PCNA ubiquitylation plays a key role in the tolerance to DNA damage in eukaryotes. Although the evolutionary conserved mechanism of PCNA ubiquitylation is well understood, the deubiquitylation of ubPCNA remains poorly characterized. Here, we show that the histone H2B(K123) ubiquitin protease Ubp10 also deubiquitylates ubPCNA in Saccharomyces cerevisiae. Our results sustain that Ubp10-dependent deubiquitylation of the sliding clamp PCNA normally takes place during S phase, likely in response to the simple presence of ubPCNA. In agreement with this, we show that Ubp10 forms a complex with PCNA in vivo. Interestingly, we also show that deletion of UBP10 alters in different ways the interaction of PCNA with DNA polymerase ?-associated protein Rev1 and with accessory subunit Rev7. While deletion of UBP10 enhances PCNA-Rev1 interaction, it decreases significantly Rev7 binding to the sliding clamp. Finally, we report that Ubp10 counteracts Rad18 E3-ubiquitin ligase activity on PCNA at lysine 164 in such a manner that deregulation of Ubp10 expression causes tolerance impairment and MMS hypersensitivity.

| S-EPMC3400564 | biostudies-literature

Mismatch Recognition by Saccharomyces cerevisiae Msh2-Msh6: Role of Structure and Dynamics.

Project description:The mismatch repair (MMR) pathway maintains genome integrity by correcting errors such as mismatched base pairs formed during DNA replication. In MMR, Msh2-Msh6, a heterodimeric protein, targets single base mismatches and small insertion/deletion loops for repair. By incorporating the fluorescent nucleoside base analog 6-methylisoxanthopterin (6-MI) at or adjacent to a mismatch site to probe the structural and dynamic elements of the mismatch, we address how Msh2-Msh6 recognizes these mismatches for repair within the context of matched DNA. Fluorescence quantum yield and rotational correlation time measurements indicate that local base dynamics linearly correlate with Saccharomyces cerevisiae Msh2-Msh6 binding affinity where the protein exhibits a higher affinity (KD ≤ 25 nM) for mismatches that have a significant amount of dynamic motion. Energy transfer measurements measuring global DNA bending find that mismatches that are both well and poorly recognized by Msh2-Msh6 experience the same amount of protein-induced bending. Finally, base-specific dynamics coupled with protein-induced blue shifts in peak emission strongly support the crystallographic model of directional binding, in which Phe 432 of Msh6 intercalates 3' of the mismatch. These results imply an important role for local base dynamics in the initial recognition step of MMR.

| S-EPMC6747400 | biostudies-literature

Photo-activatable Ub-PCNA probes reveal new structural features of the Saccharomyces cerevisiae Polη/PCNA complex.

Project description:The Y-family DNA polymerase η (Polη) is critical for the synthesis past damaged DNA nucleotides in yeast through translesion DNA synthesis (TLS). TLS is initiated by monoubiquitination of proliferating cell nuclear antigen (PCNA) and the subsequent recruitment of TLS polymerases. Although individual structures of the Polη catalytic core and PCNA have been solved, a high-resolution structure of the complex of Polη/PCNA or Polη/monoubiquitinated PCNA (Ub-PCNA) still remains elusive, partly due to the disordered Polη C-terminal region and the flexibility of ubiquitin on PCNA. To circumvent these obstacles and obtain structural insights into this important TLS polymerase complex, we developed photo-activatable PCNA and Ub-PCNA probes containing a p-benzoyl-L-phenylalanine (pBpa) crosslinker at selected positions on PCNA. By photo-crosslinking the probes with full-length Polη, specific crosslinking sites were identified following tryptic digestion and tandem mass spectrometry analysis. We discovered direct interactions of the Polη catalytic core and its C-terminal region with both sides of the PCNA ring. Model building using the crosslinking site information as a restraint revealed multiple conformations of Polη in the polymerase complex. Availability of the photo-activatable PCNA and Ub-PCNA probes will also facilitate investigations into other PCNA-containing complexes important for DNA replication, repair and damage tolerance.

| S-EPMC8450101 | biostudies-literature

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

Project description: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.

| S-EPMC4674293 | biostudies-literature

Chimeric Saccharomyces cerevisiae Msh6 protein with an Msh3 mispair-binding domain combines properties of both proteins.

Project description:Msh2-Msh3 and Msh2-Msh6 are two partially redundant mispair-recognition complexes that initiate mismatch repair in eukaryotes. Crystal structures of the prokaryotic homolog MutS suggest the mechanism by which Msh6 interacts with mispairs because key mispair-contacting residues are conserved in these two proteins. Because Msh3 lacks these conserved residues, we constructed a series of mutants to investigate the requirements for mispair interaction by Msh3. We found that a chimeric protein in which the mispair-binding domain (MBD) of Msh6 was replaced by the equivalent domain of Msh3 was functional for mismatch repair. This chimera possessed the mispair-binding specificity of Msh3 and revealed that communication between the MBD and the ATPase domain is conserved between Msh2-Msh3 and Msh2-Msh6. Further, the chimeric protein retained Msh6-like properties with respect to genetic interactions with the MutL homologs and an Msh2 MBD deletion mutant, indicating that Msh3-like behaviors beyond mispair specificity are not features controlled by the MBD.

| S-EPMC1904149 | biostudies-literature

Saccharomyces cerevisiae Msh2-Msh6 DNA binding kinetics reveal a mechanism of targeting sites for DNA mismatch repair.

Project description:The DNA mismatch repair system (MMR) identifies replication errors and damaged bases in DNA and functions to preserve genomic integrity. MutS performs the task of locating mismatched base pairs, loops and lesions and initiating MMR, and the fundamental question of how this protein targets specific sites in DNA is unresolved. To address this question, we examined the interactions between Saccharomyces cerevisiae Msh2-Msh6, a eukaryotic MutS homolog, and DNA in real time. The reaction kinetics reveal that Msh2-Msh6 binds a variety of sites at similarly fast rates (k (ON) approximately 10(7) M(-1) s(-1)), and its selectivity manifests in differential dissociation rates; e.g., the protein releases a 2-Aminopurine:T base pair approximately 90-fold faster than a G:T mismatch. On releasing the 2-Ap:T site, Msh2-Msh6 is able to move laterally on DNA to locate a nearby G:T site. The long-lived Msh2-Msh6.G:T complex triggers the next step in MMR--formation of an ATP-bound clamp--more effectively than the short-lived Msh2-Msh6.2-Ap:T complex. Mutation of Glu in the conserved Phe-X-Glu DNA binding motif stabilizes Msh2-Msh6(E339A).2-Ap:T complex, and the mutant can signal 2-Ap:T repair as effectively as wild-type Msh2-Msh6 signals G:T repair. These findings suggest a targeting mechanism whereby Msh2-Msh6 scans DNA, interrogating base pairs by transient contacts and pausing at potential target sites, and the longer the pause the greater the likelihood of MMR.

| S-EPMC2818965 | biostudies-literature

Access to PCNA by Srs2 and Elg1 Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae.

Project description:During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances, the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA-damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here, we show that in addition to translesion synthesis and strand-transfer DDT mechanisms, an alternative repair mechanism ("salvage recombination") that copies information from the sister chromatid is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1.IMPORTANCE PCNA, the ring that encircles DNA maintaining the processivity of DNA polymerases, is modified by ubiquitin and SUMO. Whereas ubiquitin is required for bypassing lesions through the DNA damage tolerance (DDT) pathways, we show here that SUMOylation represses another pathway, salvage recombination. The Srs2 helicase is recruited to SUMOylated PCNA and prevents the salvage pathway from acting. The pathway can be induced by overexpressing the PCNA unloader Elg1, or the homologous recombination protein Rad52. Our results underscore the role of PCNA modifications in controlling the various bypass and DNA repair mechanisms.

| S-EPMC7403780 | biostudies-literature

Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

Project description:Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms.

| S-EPMC4334453 | biostudies-literature

Individual lysine acetylations on the N terminus of Saccharomyces cerevisiae H2A.Z are highly but not differentially regulated.

Project description:The multi-functional histone variant Htz1 (Saccharomyces cerevisiae H2A.Z) is acetylated on up to four N-terminal lysines at positions 3, 8, 10, and 14. It has thus been posited that specific acetylated forms of the histone could regulate distinct roles. Antibodies against Htz1-K8(Ac), -K10(Ac), and -K14(Ac) show that all three modifications are added by Esa1 acetyltransferase and removed by Hda1 deacetylase. Completely unacetylatable htz1 alleles exhibit widespread interactions in genome scale genetic screening. However, singly mutated (e.g. htz1-K8R) or singly acetylable (e.g. the triple mutant htz1-K3R/K10R/K14R) alleles show no significant defects in these analyses. This suggests that the N-terminal acetylations on Htz1 are internally redundant. Further supporting this proposal, each acetylation decays with similar kinetics when Htz1 transcription is repressed, and proteomic screening did not find a single condition in which one Htz1(Ac) was differentially regulated. However, whereas the individual acetylations on Htz1 may be redundant, they are not dispensable. Completely unacetylatable htz1 alleles display genetic interactions and phenotypes in common with and distinct from htz1Δ. In addition, each Htz1 N-terminal lysine is deacetylated by Hda1 in response to benomyl and reacetylated when this agent is removed. Such active regulation suggests that acetylation plays a significant role in Htz1 function.

| S-EPMC3000967 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data