'Columbia University Libraries/Information Services'
Doi
Abstract
Life on Earth relies on a set of instructions encoded within an organism’s genome that is passed along from one generation to the next. Inherent to this mechanism of propagation is the need to copy the genetic material before passing it along to the progeny. Errors in this process coupled with stochastic damage will inevitably lead to changes in these instructions and may result in a reduction of fitness or even death of an individual. Yet, these same changes are also responsible for the adaptation mandated by our dynamic environment. Thus, there exists a delicate balance between maintenance and alteration of genetic material that is embodied to a large part at the various intersections of DNA replication, recombination and repair. Homologous recombination (HR) has been well studied and found to play vital roles in many cellular processes from the repair of the harrowing double-stranded break, the restart of a stalled or collapsed replication fork, as well as proper chromosome segregation during meiosis, all with the goal of striking this delicate balance. And yet, while HR is incumbent for the fitness of an organism, if left unchecked this same process can become detrimental by preventing better suited DNA repair pathways, permanently arresting cell cycle progression and creating some of the very problems it was meant to address such as aneuploidy or cancer. Despite a wealth of knowledge, the precise regulatory mechanisms remain an active area of research as they provide likely targets to combat these persistent diseases. Motor proteins that translocate along DNA have been particularly compelling and elusive due to their transitory nature, as well as the inevitability of collisions with bound protein(s) or nucleic acid structures that are likely regulated intermediates in the process. The yeast Srs2 helicase/translocase has long been regarded as the prototypical “anti-recombinase” as it has been shown to dismantle the Rad51 presynaptic filament, but also displays contradictory pro-recombinase functions. In vivo studies of Srs2 have been hampered by its involvement in multiple bioprocesses beyond recombination, while bulk in vitro approaches often produce conflicting results. Recent single molecule imaging of these players has shed light onto their involvement in the regulation of the various stages of the canonical pathway of HR. The Greene laboratory has developed ssDNA curtains to study the pre-synaptic filament and shown that Rad51-ssDNA filaments can create bonafide D-loop intermediates that would be incapable of repair and thus represent a toxic intermediate. These structures persist far longer than the entire process of DSBR in vivo and led us to hypothesize that motor proteins would be a key regulatory element to dismantle improperly paired intermediates for redistribution of the bound proteins and reengagement of the homology search process. Here I extend the use of ssDNA curtains to study Srs2 as it assembles into multimeric complexes to perform long-range disruption of various pre- and post-synaptic filament assemblies that include replication protein A (RPA), Rad51, Rad52, and D-loops. For the first time, direct observation of Srs2 translocating over RPA filaments is provided and shows these proteins are efficiently removed by Srs2. By including Rad52 on the RPA filament, I offer a refined model of the contradictory pro- and anti-recombinase activities of Srs2 through its antagonism of the single-strand annealing pathway in favor of HR. Additionally, Srs2 was found to initiate Rad51 disruption at breaks in the continuity of the filament marked by the persistence of replication protein A (RPA), Rad52, or the presence of an improper D-loop intermediate, the latter of which is efficiently disrupted before continuing translocation. In contrast to the prevailing model, we demonstrate that direct interaction between Srs2 and Rad51 is not necessary for long-range Rad51 clearance. These findings offer insights into the dynamic regulation of crucial HR intermediates by Srs2 and demonstrate that sub-nuclear concentrations of these proteins may be a likely driver for their activities
