SUMMARY MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSβ) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.

We present an approach for quantitative analysis of changes in the composition and phosphorylation of protein complexes by MS. It is based on a new class of stable isotope-labeling reagent, the amine-reactive isotope tag (N-isotag), for specific and quantitative labeling of peptides following proteolytic digestion of proteins. Application of the N-isotag method to the analysis of Rad53, a DNA damage checkpoint kinase in Saccharomyces cerevisiae, led to the identification of dynamic associations between Rad53 and the nuclear transport machinery, histones, and chromatin assembly proteins in response to DNA damage. Over 30 phosphorylation sites of Rad53 and its associated proteins were identified and quantified, and they showed different changes in phosphorylation in response to DNA damage. Interestingly, Ser 789 of Rad53 was found to be a major initial phosphorylation site, and its phosphorylation regulates the Rad53 abundance in response to DNA damage. Collectively, these results demonstrate that N-isotag-based quantitative MS is generally applicable to study dynamic changes in the composition of protein complexes and their phosphorylation patterns in a site-specific manner in response to different cell stimuli. Molecular & Cellular Proteomics 4:1358 -1369, 2005.MS is a central tool in proteomics and is widely used to study protein complexes and their post-translational modifications. For stably associated protein complexes, it is possible to carry out a multistep purification to obtain highly purified proteins for MS analysis (1, 2). However, numerous biologically important protein-protein interactions are dynamic and are regulated by reversible protein modifications such as phosphorylation. It is therefore important to develop new tools for quantitative analysis of dynamic changes in protein complexes under different cell stimuli. Given the nature of dynamic protein-protein interactions, rapid single step protein purification should help to preserve the interaction (3, 4). However, such purification procedures often result in higher levels of contaminating proteins, which may obscure the observation of less abundant yet specifically associated proteins by conventional in-gel digestion and MS approaches. Recently, the ICAT method was used to identify specific interacting proteins in Pol II general transcriptional complexes, thus demonstrating the potential of quantitative analysis of protein complexes by stable isotope labeling (5). However, only cysteine-containing peptides were labeled by ICAT; thus it is not applicable to quantify noncysteine-containing proteins and various protein modifications.MS has also been used to identify phosphorylation sites of proteins (6). Many signaling proteins are subjected to dynamic phosphorylation at many sites, thus understanding the biological function of phosphorylation requires analysis of phosphorylation in a site-specific and quantitative fashion (7). Existing approaches for quantitative analysis of protein phosphorylation include the use of heavy amino acids in cell cu...

The Rrm3 DNA helicase of Saccharomyces cerevisiae interacts with proliferating cell nuclear antigen and is required for replication fork progression through ribosomal DNA repeats and subtelomeric and telomeric DNA. Here, we show that rrm3 srs2 and rrm3 sgs1 mutants, in which two different DNA helicases have been inactivated, exhibit a severe growth defect and undergo frequent cell death. Cells lacking Rrm3 and Srs2 arrest in the G 2 /M phase of the cell cycle with 2N DNA content and frequently contain only a single nucleus. The phenotypes of rrm3 srs2 and rrm3 sgs1 mutants were suppressed by disrupting early steps of homologous recombination. These observations identify Rrm3 as a new member of a network of pathways, involving Sgs1 and Srs2 helicases and Mus81 endonuclease, suggested to act during repair of stalled replication forks.