The efficient repair of DNA double strand breaks (DSBs) is critical to the maintenance of genome stability and cell survival. In mammalian cells, DSBs can be repaired via a high fidelity pathway known as homologous recombination (HR). Two key enzymes involved in mitotic HR are the recombinase Rad51 and the Swi2/Snf2 motor protein and recombination mediator, Rad54. These enzymes act together with Rad51 forming a large helical, nucleoprotein filament that is the scaffolding on which HR takes place. Rad54 binds to these filaments and stimulates the function of Rad51 in a complex fashion. In meiotic recombination, the primary recombinase is Dmc1 and its activity is stimulated by the meiotic mediator Hop2-Mnd1.

To understand how these mediators stimulate the activity of their respective recombinases, we directly visualized these enzymes acting on single molecules of duplex DNA in real-time using optical tweezers and video, fluorescence-microscopy. The results reveal that both proteins facilitate capture of dsDNA into recombinase filaments and then proceed to collapse the captured DNA around the filament. DNA collapse changes the dynamics of the homology search, increasing the overall efficiency. Interestingly, the mechanism by which each mediator achieves DNA collapse and thus stimulation of their respective recombinase is different. Rad54 combines ATP hydrolysis-coupled dsDNA translocation with a novel dsDNA capture or cross-bridging activity. Hop2-mnd1 which does NOT bind ATP, utilizes rapid condensation and decondesation activities to stimulate different stages of recombination.

Collagen fiber assembly affects many physiological processes and is tightly controlled by collagen binding proteins. However to what extent membrane bound vs. cell secreted collagen binding proteins affect collagen fibrillogenesis is not well-understood. In our current study, we demonstrate that the membrane anchored extracellular domain (ECD) of the collagen receptor Discoidin Domain Receptor2 (DDR2) inhibits fibrillogenesis of collagen endogenously secreted by the cells. These results elucidate a novel functional role of the DDR2 ECD. However since soluble forms of DDR1 and DDR2 containing its extracellular domain (ECD) are known to naturally exist in the extracellular matrix, we further investigate if soluble DDR ECDs may have a functional role in modulating collagen fibrillogenesis. In this study, we created mouse osteoblast cell lines stably expressing membrane-anchored DDR2 ECD and mouse osteoblast cell lines stably secreting DDR1 or DDR2 ECD as soluble proteins. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays were used to demonstrate that DDR ECD expression reduced the rate and quantity of collagen deposition and induced significant changes in fiber morphology and matrix mineralization. Collectively, our studies advance our understanding of DDR receptors as powerful regulators of collagen deposition in the ECM and elucidate their multifaceted role in ECM remodeling.

In the heart, Ca²⁺ released from the intracellular Ca²⁺ storage site, the sarcoplasmic reticulum (SR), is the principal determinant of cardiac contractility. SR Ca²⁺ release is controlled by dedicated molecular machinery, composed of the cardiac ryanodine receptor (RyR2) and a number of accessory proteins, including FKBP12.6, calsequestrin (CASQ2), triadin (TRD) and junctin (JN). Acquired and genetic defects in the components of the release channel complex result in a spectrum of abnormal Ca²⁺ release phenotypes ranging from arrhythmogenic spontaneous Ca²⁺ releases and Ca²⁺ alternans to the uniformly diminished systolic Ca²⁺ release characteristic of heart failure. In my presentation I will discuss the relationships between abnormal SR Ca²⁺ release and various cardiac disease phenotypes, including, arrhythmias and heart failure, and consider SR Ca²⁺ release as a potential therapeutic target.