Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damageinduced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damageinduced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.Cellular surveillance of genome integrity and repair of DNA damage are essential processes that ensure proper development and survival of all organisms. DNA double-strand breaks (DSBs) 2 are a particularly deleterious form of genome damage and occur following exposure of cells to exogenous mutagens as well as during normal metabolic processes, e.g. antigen receptor gene rearrangement, restart of stalled replication forks, formation of meiotic DNA crossovers, etc. (1, 2). Mammalian cells use two distinct mechanisms for repair of DSBs, non-homologous end joining and homologous recombination (HR). Processes requiring imprecise DNA repair, such as the creation of antibody diversity, exploit the error-prone non-homologous end joining mechanism. In contrast, HR is an error-free DNA repair pathway and is critical for avoidance of unwanted genetic changes during the meiotic exchange of information between paternal and maternal alleles and for error-free repair of broken chromosomes (3). Rad51 is the central enzymatic component of HR. Upon its regulated recruitment to sites of DNA breaks, Rad51 forms a nucleoprotein filament by polymerizing onto single-stranded DNA at the processed break. This filament catalyzes DNA strand exchange with an undamaged sister chromatid or homologous chromosome, which serves as a template for the restoration of missing genetic information (3, 4).Visible nuclear Rad51 clusters, or foci, form during S-phase and appear to localize to sites of replicating DNA (5-7). A dramatic increase in the number and size of nuclear Rad51 foci is a hallmark of the early cellular response to genomic insult (7-10). The appearance of DNA damage-induced nuclear Rad51 foci is blocked in cells with deficiencies in several HR-related proteins, including Brca2 ...

The bacterial RecA protein participates in a remarkably diverse set of functions, all of which are involved in the maintenance of genomic integrity. RecA is a central component in both the catalysis of recombinational DNA repair and the regulation of the cellular SOS response. Despite the mechanistic differences of its functions, all require formation of an active RecA/ATP/DNA complex. RecA is a classic allosterically regulated enzyme, and ATP binding results in a dramatic increase in DNA binding affinity and a cooperative assembly of RecA subunits to form an ordered, helical nucleoprotein filament. The molecular events that underlie this ATP-induced structural transition are becoming increasingly clear. This review focuses on descriptions of our current understanding of the molecular design and allosteric regulation of RecA. We present a comprehensive list of all published recA mutants and use the results of various genetic and biochemical studies, together with available structural information, to develop ideas regarding the design of RecA functional domains and their catalytic organization.

The human RAD52 protein plays an important role in the earliest stages of chromosomal double-strand break repair via the homologous recombination pathway. Individual subunits of RAD52 self-associate into rings that can then form higher order complexes. RAD52 binds to double-strand DNA ends, and recent studies suggest that the higher order self-association of the rings promotes DNA end-joining. Earlier studies defined the selfassociation domain of RAD52 to a unique region in the N-terminal half of the protein. Here we show that there are in fact two experimentally separable self-association domains in RAD52. The N-terminal self-association domain mediates the assembly of monomers into rings, and the previously unidentified domain in the C-terminal half of the protein mediates higher order self-association of the rings.The repair of double-strand breaks in chromosomal DNA is of critical importance for the maintenance of genomic integrity. In Saccharomyces cerevisiae, genes of the RAD52 epistasis group, RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2, were identified initially by the sensitivity of mutants to ionizing radiation (1, 2). These genes have been implicated in an array of recombination events including mitotic and meiotic recombination as well as doublestrand break repair. RAD52 mutants show the most severe pleiotropic defects suggesting a critical role for the protein in homologous recombination and double-strand break repair (2). The importance of specific protein-protein interactions in the catalysis of homologous recombination is suggested by studies demonstrating specific contacts and functional interactions between Rad52p and a number of proteins involved in recombination including Rad51p (3-8), which catalyzes homologous pairing and strand exchange, and replication factor A (RPA) 1 (8 -10), a heterotrimeric single-stranded DNA binding protein (11).Studies of the equivalent human proteins have identified similar interactions between the RAD52, RAD51, and replication protein A proteins (12-17). Based on a series of proteinprotein interaction assays (15,16,18) and DNA binding studies 2 (16), a domain map of RAD52 was proposed by Park et al. (16) (see Fig. 1). The determinants of self-association were proposed to exist exclusively within a region defined by residues 65-165, a result supported by recent studies of several isoforms of RAD52 (19). Electron microscopy (EM) studies of Rad52p and RAD52 have revealed formation of ring-shaped structures (9 -13 nm in diameter), as well as higher order aggregates (9,12,20). Stasiak et al. (21) performed image analyses of negatively stained electron micrographs and determined that the 10-nm RAD52 rings are composed of seven subunits. Scanning transmission electron microscopy (STEM) analysis indicated a mean mass of 330 Ϯ 59 kDa supporting a heptameric ring-shaped RAD52 structure (21). Recent studies show that RAD52 binds to double-stranded DNA ends as an aggregated complex (20). These end-binding complexes were amorphous in shape and ranged in ...

DNA double-strand breaks (DSBs) caused by cellular exposure to genotoxic agents or produced by inherent metabolic processes initiate a rapid and highly coordinated series of molecular events resulting in DNA damage signaling and repair. Phosphorylation of histone H2AX to form ␥-H2AX is one of the earliest of these events and is important for coordination of signaling and repair activities. An intriguing aspect of H2AX phosphorylation is that ␥-H2AX spreads a limited distance up to 1-2 Mbp from the site of a DNA break in mammalian cells. However, neither the distribution of H2AX throughout the genome nor the mechanism that defines the boundary of ␥-H2AX spreading have yet been described. Here, we report the identification of previously undescribed H2AX chromatin structures by successfully applying 4Pi microscopy to visualize endogenous nuclear proteins. Our observations suggest that H2AX is not distributed randomly throughout bulk chromatin, rather it exists in distinct clusters that themselves are uniformly distributed within the nuclear volume. These data support a model in which the size and distribution of H2AX clusters define the boundaries of ␥-H2AX spreading and also may provide a platform for the immediate and robust response observed after DNA damage.␥-H2AX ͉ H2AX chromatin clusters ͉ super-resolution 4Pi microscopy ͉ chromatin response to DNA damage ͉ 3D quantification of chromatin structures M aintenance of genome integrity is critical for organism development and survival, and higher organisms have evolved sophisticated mechanisms for detection and repair of chromosome breaks. DNA damage results in the rapid and coordinated action of various pathways, including activation of cell cycle checkpoints (1, 2), histone modification near the site of the break (3, 4), and recruitment of chromatin remodeling enzymes (4-6), cohesins (7-9), and DNA repair proteins (1, 2, 10). The significance of these molecular processes is highlighted by the fact that defects in many are associated with an increased risk of cancer and developmental and immunologic abnormalities (1). Important insights into the positioning of nuclear signaling and repair proteins and their response to various types and levels of genomic insults have been achieved by using immunofluorescence methods (10-12). However, it has been impossible to distinguish f luorescent signals in a threedimensional (3D) environment that are closer together than 500-800 nm in distance given the depth resolution limits of current light microscopes. With Ϸ100-nm resolution along the optic axis (z axis), 4Pi microscopy (13, 14) provides a significant increase in resolution and has allowed more defined images of cellular structures such as microtubules, mitochondria, or the Golgi apparatus (15). However, until now, imaging of endogenous nuclear proteins had not been achieved (a comparison of 4Pi vs. confocal is described in Materials and Methods). In this study, we describe the successful use of 4Pi microscopy to visualize endogenous nuclear proteins. By applying previo...

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