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The Rad51 nucleoprotein filament mediates DNA strand exchange, a key step of homologous recombination. This activity is stimulated by replication protein A (RPA), but only when RPA is introduced after Rad51 nucleoprotein filament formation. In contrast, RPA inhibits Rad51 nucleoprotein complex formation by prior binding to single-stranded DNA (ssDNA), but Rad52 protein alleviates this inhibition. Here we show that Rad51 filament formation is simultaneous with displacement of RPA from ssDNA. This displacement is initiated by a rate-limiting nucleation of Rad51 protein onto ssDNA complex, followed by rapid elongation of the filament. Rad52 protein accelerates RPA displacement by Rad51 protein. This acceleration probably involves direct interactions with both Rad51 protein and RPA. Detection of a Rad52-RPA-ssDNA co-complex suggests that this co-complex is an intermediate in the displacement process.

RAD54is an important member of the RAD52group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.

Protein-promoted DNA strand exchange requires formation of an active presynaptic complex between the DNA-pairing protein and single-stranded DNA (ssDNA). Formation of such a contiguous filament is stimulated by a ssDNA-binding protein. Here, the effects of replication protein A (RPA) on presynaptic complex formation and DNA strand exchange activities of Rad51 protein were examined. Presynaptic complex formation was assessed by measuring ATP hydrolysis. With ϕX174 ssDNA, the ATPase activity of Rad51 protein is stimulated ∼1.4-fold by RPA, provided that Rad51 protein is in excess of the ssDNA concentration; otherwise, RPA inhibits ATPase activity. In contrast, with ssDNA devoid of secondary structure (poly(dT), poly(dA), poly(dI), and etheno-M13 DNA), RPA does not stimulate the already elevated ATPase activity of Rad51 protein, but inhibits activity at low Rad51 protein concentrations. These results suggest that Rad51 protein and RPA exclude one another from ssDNA by competing for the same binding sites and that RPA exerts its effect on presynaptic complex formation by eliminating secondary structure to which Rad51 protein is bound nonproductively. DNA strand exchange catalyzed by Rad51 protein is also greatly stimulated by RPA. The optimal stoichiometry for stimulation is ∼20-30 nucleotides of ssDNA/RPA heterotrimer. The ssDNA-binding protein of Escherichia colican substitute for RPA, showing that the role of RPA is not specific. We conclude that RPA affects both presynaptic complex formation and DNA strand exchange via changes in DNA structure, employing the same mechanism used by the ssDNA-binding protein to effect change in E. coliRecA protein activity.

•Repeated sunlight exposure and incubations induced mutations in vitro.•Photolyase-independent photoreversal of CPD is involved in a mutational process.•Pol η-mediated mutagenesis requires cytosine deamination in pyrimidine dimers.•Sunlight-induced mutation spectra were similar to a cancer mutational signature.•Pol ι produces distinct mutation spectra on UV-irradiated DNA.

We fabricated blue (${\sim}450$ nm), blue-green (${\sim}500$ nm), and green (${\sim}525$ nm) light-emitting diodes (LEDs) of different dislocation densities (DD) and characterized their internal quantum efficiency (IQE). The IQE of the blue LEDs fabricated using GaN substrate exceeded 90% (DD: ${\sim}10^{6}$ cm-2), however, when we used a GaN-on-sapphire substrate (DD: ${\sim}10^{8}$ cm-2), IQE was limited to ${\sim}60$%. Droop was reduced by use of the GaN substrate. The junction temperature of the GaN-on-sapphire substrate was found to be ${\sim}200$ °C although the junction temperature of the GaN substrate was ${\sim}50$ °C when a forward current of 100 A/cm2was driven. A lowering of IQE in green LEDs to ${\sim}60$% was observed, even though we used a low-dislocation-density substrate [DD: $(1{\mbox{--}}2)\times 10^{7}$ cm-2]. The junction temperature of blue-green and green LEDs was about 100 °C when a forward current of 177 A/cm2was driven, which indicated that junction temperature is not a major factor for IQE suppression in green LEDs.

Expression of the Rep protein of colicin E2 plasmid is negatively controlled at a post-transcriptional step by a plasmid-coded RNA (RNAI). RNAI is complementary to the 5' nontranslated region of the Rep mRNA and can be folded into two stem-loop structures. Several cop mutations that increased the copy number of the plasmid have been mapped in the RNAI coding region. Here we demonstrated that RNAI and the Rep mRNA rapidly form a stable complex under near physiological conditions. The cop mutations drastically reduced the binding rate. The reduction correlated to the in vivo phenotypes of the cop mutant plasmids, indicating that binding of RNAI to the Rep mRNA is involved in the regulation of the Rep protein expression. Binding properties of RNAI and the Rep mRNA with the cop mutations and those with various extents of deletion suggested that the initial interaction of the two RNAs occurs between the loop portions of the larger of the two stem-loop structures. RNAI does not completely sequester the putative S/D region, even when the two RNAs form a complete duplex. Possible mechanisms of the inhibition of the rep gene expression by binding of RNAI to the Rep mRNA under such a situation were discussed.