Rad52 plays essential roles in homology-dependent doublestrand break repair. Various studies have established the functions of Rad52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of Rad52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within Rad52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the Rad52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that Rad52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that Rad52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.The repair of DSBs 4 is a critical process by which cells maintain genome integrity. In eukaryotic cells these breaks are repaired by homologous recombination (HR) and non-homologous end joining. In the homologous end joining pathway, broken DNA ends are rejoined by religation or annealing of short common sequences (microhomologies) near their ends.Because homologous end joining lacks a mechanism to reject erroneous pairings that occur by chance, recessed DNA, chromosome rearrangements, and mutations are possible outcomes of this repair pathway (1). By contrast, the HR pathway achieves high accuracy in DSB repair by utilizing mechanisms for a DNA homology search. In this pathway the ends of the DSBs are resected to generate 3Ј single-stranded tails. The singlestranded region then either invades an undamaged homologous duplex DNA (strand invasion) or pairs with a complementary single strand (single-strand annealing). The importance of this pathway is underscored by the high conservation, from yeast to humans, of the factors that catalyze HR (2).Rad52 is one of the key players in the yeast HR pathway, and its homologs have been found in higher eukaryotes. In yeast, Rad52 is essential in both the RAD51-dependent and -independent HR pathways and functions in several homology-dependent DSB repair events, including gene conversion, breakinduced replication, and recombination between inverted repeats (3). Several biochemical studies have established that Rad52 functions as a mediator in RAD51-dependent HR pathways. These findings include facilitating the assembly of the Rad51 recombinase on replication protein A-coated ssDNA (4, 5) and stimulating the DNA strand exchange activity of the Rad51 recombinase (6 -9). In vitro studies have also suggested more direct roles of Rad52 in HR. Rad52 catalyzes the annealing of complementary ssDNA and promotes D-loop formation and DNA strand exchange (10 -14). The strand invasion promoted by Rad52 may be relevant to some RAD51-independent HR events such as break-induced replication, in...

The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosisspecific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strandexchange activity of Dmc1 by stabilizing the Dmc1-single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.

The assembly state of enzymes is gaining interest as a mechanism for regulating the function of enzymes in living cells. One of the current topics in enzymology is the relationship between enzyme activity and the assembly state due to liquid–liquid phase separation. In this study, we demonstrated enzyme activation via the formation of enzyme assemblies using L-lactate oxidase (LOX). LOX formed hundreds of nanometer-scale assemblies with poly-L-lysine (PLL). In the presence of ammonium sulfate, the LOX-PLL clusters formed micrometer-scale liquid droplets. The enzyme activities of LOX in clusters and droplets were one order of magnitude higher than those in the dispersed state, owing to a decrease in KM and an increase in kcat. Moreover, the clusters exhibited a higher activation effect than the droplets. In addition, the conformation of LOX changed in the clusters, resulting in increased enzyme activation. Understanding enzyme activation and assembly states provides important information regarding enzyme function in living cells, in addition to biotechnology applications.

Liquid droplets formed by liquid-liquid phase separation are attracting attention as functional states of proteins in living cells. Liquid droplets are thought to activate enzymatic reactions by assembling the required molecules. Thus, liquid droplets usually increase the affinity of an enzyme to its substrates, leading to decreased KM values. In this study, we demonstrate a new mechanism of enzyme activation in the droplets using Llactate oxidase (LOX). In the presence of poly-L-lysine (PLL), LOX formed droplets with diameters of hundreds of nanometers to tens of micrometers, stabilized by electro-static interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold decrease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increasing kcat by the formation of the liquid droplet. Interestingly, the conformation of LOX changed in the liquid droplet, probably leading to increased kcat value. Understanding enzyme activation in the droplets provides essential information about enzyme function in living cells in addition to biotechnology applications.