Showing papers by "Emilios K. Dimitriadis published in 2000"
TL;DR: The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding interactions involvingbeta-Pol.
Abstract: XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase beta (beta-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain-domain interaction in the XRCC1-beta-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I-linker-BRCT-II C-terminal fragment and the linker-BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact beta-Pol and the beta-Pol 31 kDa domain. The XRCC1-NTD(1-183)(residues 1-183) was found to bind beta-Pol, the beta-Pol 31 kDa domain and the beta-Pol C-terminal palm-thumb (residues 140-335), and the interaction was further localized to XRCC1-NTD(1-157)(residues 1-157). The XRCC1-NTD(1-183)-beta-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36-355 and residues 1-159, were found to interact with beta-Pol, the beta-Pol 31 kDa domain, and the beta-Pol C-terminal thumb-only domain polypeptides expressed from the respective beta-Pol constructs. Neither the XRCC1-NTD(1-159), nor the XRCC1(36-355)polypeptide was found to interact with a beta-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75-212) showed no interaction with beta-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD(1-183)was found to bind beta-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K(d) of 0.4-2.4 microM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD(1-159)and beta-Pol that is of an affinity comparable to other binding interactions involving beta-Pol.
107 citations
TL;DR: This chapter describes the optimal data analysis, using transmitted light intensities, in analytical ultracentrifuge, and provides assumptions for the correct model and the adequate data points.
Abstract: Publisher Summary This chapter describes the optimal data analysis, using transmitted light intensities, in analytical ultracentrifuge. Analytical ultracentrifugation machines, such as the Beckman XL-A, may be equipped with absorbance optics that is essentially a dual-beam spectrophotometer, with a xenon lamp and a monochromator. To investigate the state of the solution, solute behavior models are assumed and corresponding mathematical models for the expected concentration profiles are written. The suitability of the assumed models is then tested by attempting to curve fit the mathematical models to the collected absorbance data. The chapter provides assumptions for the correct model and the adequate data points are: (1) the data are independent of each other, (2) there is no other systematic error, and (3) the noise probability distribution is Gaussian. Usually, quantities, such as molecular masses and association constants or sedimentation and diffusion coefficients, are used as the fitting parameters, for modeling sedimentation equilibrium or velocity data, respectively.
2 citations