Poly(ADP-ribosyl)ation associated changes in CTCF-chromatin binding and gene expression in breast cells

CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180 kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130 kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present

The structure of a ring-opened proliferating cell nuclear antigen–replication factor C complex revealed by fluorescence energy transfer

Numerous proteins that function in DNA metabolic pathways are known to interact with the proliferating cell nuclear antigen (PCNA). The important function of PCNA in stimulating various cellular activities requires its topological linkage with DNA. Loading of the circular PCNA onto duplex DNA requires the activity of a clamp-loader [replication factor C (RFC)] complex and the energy derived from ATP hydrolysis. The mechanistic and structural details regarding PCNA loading by the RFC complex are still developing. In particular, the positive identification of a long-hypothesized structure of an open clamp–RFC complex as an intermediate in loading has remained elusive. In this study, we capture an open yeast PCNA clamp in a complex with RFC through fluorescence energy transfer experiments. We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATPγS cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm

Role of Adenosine 5‘-Triphosphate Hydrolysis in The Assembly of The Bacteriophage T4 DNA Replication Holoenzyme Complex

Steady-state and pre-steady-state rates of ATP hydrolysis by the 44/62 accessory protein were determined to elucidate the role of ATP hydrolysis in bacteriophage T4 holoenzyme complex formation. Steady-state ATPase measurements of the 44/62 protein under various combinations of 45 protein, DNA substrate, and T4 exo- polymerase indicate that although the 44/62 protein synergistically hydrolyzes ATP in the presence of 45 protein and DNA substrate, the ATPase activity of 44/62 is diminished substantially upon the formation of the holoenzyme complex. The decrease in activity is primarily in kcat while the Km for ATP is changed unsubstantially by the various combinations. Data suggest that the decrease in the rate of ATP hydrolysis upon the addition of T4 exo- polymerase in the presence of 45 protein and DNA substrate is due to formation of a stable holoenzyme complex consisting of only the 45 protein and T4 exo- polymerase in a 1:1 ratio. The 44/62 protein acts catalytically to load 45 protein onto the DNA substrate and does not remain a component of the holoenzyme complex. Pre-steady-state kinetic analysis of the ATP hydrolysis reaction catalyzed by the 44/62 protein loading the 45 protein onto the DNA substrate in the absence or presence of polymerase is biphasic, in which a burst in ATP hydrolysis precedes the steady-state rate of ATP hydrolysis. An identical burst in ATP consumption is obtained under either condition, indicating that ATP hydrolysis is not required to load polymerase into the holoenzyme complex. The data suggest one turnover of ATP at each of the four ATPase active sites of the 44/62 protein per 45 protein loaded. ATP hydrolysis by the 44/62 protein under conditions of holoenzyme complex formation is the rate-limiting step in holoenzyme complex formation. The process of holoenzyme formation appears to be identical for leading and lagging strand synthesis

Measurement of protein–ligand complex formation

Experimental approaches to detect, measure, and quantify protein–ligand binding, along with their theoretical bases, are described. A range of methods for detection of protein–ligand interactions is summarized. Specific protocols are provided for a nonequilibrium procedure pull-down assay, for an equilibrium direct binding method and its modification into a competition-based measurement and for steady-state measurements based on the effects of ligands on enzyme catalysis