Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.
DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing.
- MeSH
- apoptóza MeSH
- chromatin genetika MeSH
- helikasy RecQ genetika metabolismus MeSH
- imunoenzymatické techniky MeSH
- imunoprecipitace MeSH
- interakční proteinové domény a motivy MeSH
- kur domácí genetika MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- MCM komplex, komponenta 2 genetika metabolismus MeSH
- MCM komplex, komponenta 7 genetika metabolismus MeSH
- MCM proteiny genetika metabolismus MeSH
- messenger RNA genetika MeSH
- molekulární sekvence - údaje MeSH
- nádorové buňky kultivované MeSH
- nádory kostí genetika metabolismus patologie MeSH
- osteosarkom genetika metabolismus patologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- povrchová plasmonová rezonance MeSH
- proliferace buněk MeSH
- průtoková cytometrie MeSH
- replikace DNA * MeSH
- replikační počátek genetika MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
