Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage*

Abstract

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2–7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.

Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP

“…Samples from 3 independent mass spectrometry-IPs were subjected to trypsin digestion directly on beads and the resulting peptides were separated using a Dionex Ultimate 3000 nanoHPLC system as described in (Drissi et al, 2015). Raw data issued from the MS were processed, searched and quantified using the MaxQuant software package version 1.5.1.2 (Cox and Mann, 2008) employing the yeast W303_ALAV00000000 database (12/7/2012).…”

“…Raw data issued from the MS were processed, searched and quantified using the MaxQuant software package version 1.5.1.2 (Cox and Mann, 2008) employing the yeast W303_ALAV00000000 database (12/7/2012). The settings used for the MaxQuant analysis were as described (Drissi et al, 2015). Specific proteins were defined as those that were present in at least two mass spectrometry-IPs and identified exclusively in the TLC1-MS2 sample.…”

Human PDCD2L Is an Export Substrate of CRM1 That Associates with 40S Ribosomal Subunit Precursors

“…For stable isotope labeling in cell culture (SILAC) experiments, proteins were labeled with stable isotopes of arginine and lysine in cell culture, as previously described (32). Briefly, HEK 293-FT cells expressing GFP-or Flag-tagged versions of proteins were grown in medium containing labeled amino acids (Cambridge Isotope Laboratories).…”

“…Eluates were concentrated by using a SpeedVac and resuspended in reducing buffer (125 mM Tris-HCl [pH 8.0], 1.25% SDS, 37.5% glycerol, 60 mM DTT, 0.025% bromophenol blue). Gel electrophoresis, in-gel digestions, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and analysis of SILAC ratios were performed as described previously (32).…”

MCM7 Gene Expression versus Risk of Malignancy Index (RMI) for Prediction of Ovarian Malignancy

“…They are encoded by genes which are parts of the MCM genes from MCM 2-7 which have hexametric structure [10]. MCM proteins are involved in two critical steps in DNA synthesis: the first step is DNA replication initiation mediated by hexametric MCM complexes at replication origins, and the second step is DNA elongation mediated by MCM helicase activities [11].…”