Nucleoprotein filaments made up of Rad51 or Dmc1 recombinases, the core structures of recombination, engage in ATP-dependent DNA-strand exchange. The ability of recombinases to form filaments is enhanced by recombination factors termed 'mediators'. Here, we show that the Schizosaccharomyces pombe Swi5-Sfr1 complex, a conserved eukaryotic protein complex, at substoichiometric concentrations stimulates strand exchange mediated by Rhp51 (the S. pombe Rad51 homolog) and Dmc1 on long DNA substrates. Reactions mediated by both recombinases are completely dependent on Swi5-Sfr1, replication protein A (RPA) and ATP, although RPA inhibits the reaction when it is incubated with single-stranded DNA (ssDNA) before the recombinase. The Swi5-Sfr1 complex overcomes, at least partly, the inhibitory effect of RPA, representing a novel class of mediator. Notably, the Swi5-Sfr1 complex preferentially stimulates the ssDNA-dependent ATPase activity of Rhp51, and it increases the amounts of Dmc1 bound to ssDNA.

DNA polymerase (Pol) is an error-prone DNA polymerase involved in translesion DNA synthesis. Pol consists of two subunits: the catalytic REV3, which belongs to B family DNA polymerase, and the noncatalytic REV7. REV7 also interacts with REV1 polymerase, which is an error-prone Y family DNA polymerase and is also involved in translesion DNA synthesis. Cells deficient in one of the three REV proteins and those deficient in all three proteins show similar phenotype, indicating the functional collaboration of the three REV proteins. REV7 interacts with both REV3 and REV1 polymerases, but the structure of REV7 or REV3, as well as the structural and functional basis of the REV1-REV7 and REV3-REV7 interactions, remains unknown. Here we show the first crystal structure of human REV7 in complex with a fragment of human REV3 polymerase (residues 1847-1898) and reveal the mechanism underlying REV7-REV3 interaction. The structure indicates that the interaction between REV7 and REV3 creates a structural interface for REV1 binding. Furthermore, we show that the REV7-mediated interactions are responsible for DNA damage tolerance. Our results highlight the function of REV7 as an adapter protein to recruit Pol to a lesion site. REV7 is alternatively called MAD2B or MAD2L2 and also involved in various cellular functions such as signal transduction and cell cycle regulation. Our results will provide a general structural basis for understanding the REV7 interaction.Large numbers of DNA lesions occur daily in every cell, and the majority of the DNA lesions stall replicative DNA polymerases. This results in the arrest of DNA replication, which causes lethal effects including genome instability and cell death. Translesion DNA synthesis (TLS)2 releases this replication blockage by replacing the stalled replicative polymerase with a DNA polymerase specialized for TLS (TLS polymerase). It is generally considered that TLS includes two steps performed by at least two types of TLS polymerases, namely inserter and extender polymerases (reviewed in Refs. 1 and 2). In the first step, the stalled replicative polymerase is switched to an inserter polymerase such as Pol, Pol, Pol, or REV1, which are classified as Y family DNA polymerases (3) and have different lesion specificity (reviewed in Refs. 4 -8), and an inserter polymerase incorporates nucleotides opposite the DNA lesion instead of the stalled replicative polymerase. In the second step, an inserter polymerase is switched to the extender polymerase DNA polymerase (Pol), and then Pol extends a few additional nucleotides before a replicative polymerase restarts DNA replication.Pol consists of the catalytic REV3 and the noncatalytic REV7 subunits. REV3 is classified as a B family DNA polymerase on the basis of the primary sequence. The catalytic activity of yeast REV3 is stimulated by yeast REV7 (9). Biochemical analysis has been done only for yeast REV3 but not mammalian REV3, because the molecular mass of human REV3 is larger (ϳ350 kDa) than that of yeast REV3 (ϳ150 kDa). Disruption of the ...

p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.Impairment of the ubiquitin-proteasome system is one of major causes of ubiquitin-positive inclusions found in various neurodegenerative diseases (1). Recent studies have identified the involvement of another degradation system, the autophagy-lysosomal pathway, in the formation of ubiquitin-positive inclusions as exemplified by the observation that autophagy-deficient mice exhibit substantial accumulation of such inclusions in tissues (2). p62/SQSTM1/A170, a multidomain protein found in ubiquitin-positive inclusions, has been shown to bind intracellular signaling factors (3-5). Accumulating evidence indicates that p62 is a receptor for ubiquitinated proteins that are targeted to the autophagosome for lysosomal degradation. Specifically, it is involved in autophagic elimination of damaged mitochondria, midbody rings, peroxisomes, and microbes (6 -10).The importance of p62 in autophagic degradation of proteins and organelles has been demonstrated by studies using tissuespecific autophagy-deficient mice. Elimination of Atg5 or Atg7, an essential gene in the formation of the autophagosome, in mouse neurons and hepatocytes resulted in toxicity accompanied by accumulation of ubiquitin-positive inclusions in the cells. In contrast, knock-out of both Atg7 and p62 (Atg7 Ϫ/Ϫ / p62 Ϫ/Ϫ ) caused a dramatic reduction in the amount of inclusions in both types of cells (2, 11). A similar result was also reported in fruit flies (12). These observations indicate that p62 is critically involved in the development of ubiquitin-positive inclusions that should be degraded via autophagy. In addition to playing a role in the autophagy-lysosomal pathway, p62 its...

Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel. Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA. This is the initial nickel receptor and mediator of the chemotactic response away from nickel. We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a "classical" periplasmic binding protein. In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed. No direct bonds are formed between the metal cation and the protein. The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site. Despite relatively weak binding, NikA is specific for nickel. Using isothermal titration calorimetry, the dissociation constant for nickel was found to be ϳ10 M and that for cobalt was approximately 20 times higher.

The modular structure of many protein families, such as β-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms.Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical β-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2-10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.protein evolution | computational protein design | self-assembly | β-propeller | protein crystallography