The eukaryotic helicase MCM2-7, is loaded by ORC, Cdc6 and Cdt1 as a double-hexamer onto replication origins. The insertion of DNA into the helicase leads to partial MCM2-7 ring closure, while ATP hydrolysis is essential for consecutive steps in pre-repl
a Detection by western blot of RPA1 and RPA2 co-precipitated by bead-bound recombinant GST, GST-SMARCAL1, or GST-MCM8IP from bacteria. b Schematic representation of full-length MCM8IP and MCM8IP with deletions in RPA1 binding motifs. The Δ1–54,153–168 mutant was renamed RBM. Shown...
pH 8, with 0.1% (v/v) Triton X-100) before adding 50 mM reduced glutathione (Sigma-Aldrich) in elution buffer. For GST pull down experiments using human cell lysates, 700 µg of proteins (soluble nucleocytoplasmic and chromatin-bound subcellular fractions) were added ...
Download: Download full-size image Figure 4. Mcm1 binds several ARSs in vitro. A, Gelcode Blue-stained SDS-polyacrylamide gel of purified, E. coli-expressed GST-Mcm1 (4 μg, lane 1) and molecular weight markers (lane 2) are shown. B–D, purified E. coli GST-Mcm1 protein was incu...
All the mutants and the Mcm4/6/7 complex for the in vitro binding assay were cloned and purified using methods identical to those described previously (22). We performed densitometric analysis for GST pulldown data by using Multi Gauge (Fujifilm) and quantified binding affinities between Cdt1 ...
Full size image Table 1 Cryo-EM data collection, refinement and validation statistics Full size table DDK engagement explains N-terminal Mcm4 phosphorylation Double hexamer loading onto origins in vitro, followed by incubation with DDK, results in activation of MCM phosphorylation as established by SDS...
HORMAD1 is a meiosis-specific protein that promotes synapsis and recombination of homologous chromosomes in meiotic prophase. Originally identified as a cancer/testis antigen, HORMAD1 is also aberrantly expressed in several cancers. However, the function
Left preparation contains GST-Mcm4p and MBP-Mcm2p; right preparation contains a C-terminal HA/HIS10 tag on all subunits except Mcm2p. (D) Competition of ATP hydrolysis by unlabeled adenosine analogs. MCM complex was assayed at 10 μM [ATP] in the absence (no comp) or presence of an ...
To confirm this, an in vitro ubiquitylation assay was performed. The KEAP1-CUL3-RBX1 complex was sufficient to ubiquitylate MCM3 (Fig. 3G). Download: Download high-res image (201KB) Download: Download full-size image FIGURE 3.. MCM3 is a KEAP1-CUL3 substrate for ubiquitylation. A, HEK...
full-length mCdt1 was mixed simultaneously with five amino-terminally truncated proteins (GSTΔN1–127, GSTΔN1–176, GSTΔN1–293, GSTΔN1–406, and GSTΔN1–478) or three carboxyl-terminally truncated proteins (GSTΔC381–557, GSTΔC180–557, and GSTΔC131–557) and incubated with DNA...