RPA interacts with Rad52 to promote meiotic crossover and noncrossover recombination.

Meiotic recombination is initiated by programmed double-strand breaks (DSBs). Studies in Saccharomyces cerevisiae have shown that, following rapid resection to generate 3' single-stranded DNA (ssDNA) tails, one DSB end engages a homolog partner chromatid and is extended by DNA synthesis, whereas the other end remains associated with its sister. Then, after regulated differentiation into crossover- and noncrossover-fated types, the second DSB end participates in the reaction by strand annealing with the extended first end, along both pathways. This second-end capture is dependent on Rad52, presumably via its known capacity to anneal two ssDNAs. Here, using physical analysis of DNA recombination, we demonstrate that this process is dependent on direct interaction of Rad52 with the ssDNA binding protein, replication protein A (RPA). Furthermore, the absence of this Rad52-RPA joint activity results in a cytologically-prominent RPA spike, which emerges from the homolog axes at sites of crossovers during the pachytene stage of the meiotic prophase. Our findings suggest that this spike represents the DSB end of a broken chromatid caused by either the displaced leading DSB end or the second DSB end, which has been unable to engage with the partner homolog-associated ssDNA. These and other results imply a close correspondence between Rad52-RPA roles in meiotic recombination and mitotic DSB repair.

Recruitment of Rec8, Pds5 and Rad61/Wapl to meiotic homolog pairing, recombination, axis formation and S-phase.

We have explored the meiotic roles of cohesin modulators Pds5 and Rad61/Wapl, in relation to one another, and to meiotic kleisin Rec8, for homolog pairing, all physically definable steps of recombination, prophase axis length and S-phase progression, in budding yeast. We show that Pds5 promotes early steps of recombination and thus homolog pairing, and also modulates axis length, with both effects independent of a sister chromatid. [Pds5+Rec8] promotes double-strand break formation, maintains homolog bias for crossover formation and promotes S-phase progression. Oppositely, the unique role of Rad61/Wapl is to promote non-crossover recombination by releasing [Pds5+Rec8]. For this effect, Rad61/Wapl probably acts to maintain homolog bias by preventing channeling into sister interactions. Mysteriously, each analyzed molecule has one role that involves neither of the other two. Overall, the presented findings suggest that Pds5's role in maintenance of sister chromatid cohesion during the mitotic prophase-analogous stage of G2/M is repurposed during meiosis prophase to promote interactions between homologs.

A Method for Physical Analysis of Recombination Intermediates in Saccharomyces cerevisiae.

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions (SEIs) and double-Holliday junctions (dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication.

Pharmacokinetic scaling of SJ-8029, a novel anticancer agent possessing microtubule and topoisomerase inhibiting activities, by species-invariant time methods.

This study examined the pharmacokinetic disposition of SJ-8029, a novel anticancer agent possessing microtubule and topoisomerase inhibiting activities, in mice, rats, rabbits and dogs after i.v. administration. The serum concentration-time curves of SJ-8029 were best described by tri-exponential equations in all these animal species. The mean Cl, V(ss) and t(1/2) were 0.3 l/h, 0.1 l and 63.2 min in mice, 1.5 l/h, 1.6 l and 247.7 min in rats, 13.8 l/h, 39.6 l and 245.9 min in rabbits, and 29.2 l/h, 44.6 l and 117.4 min in dogs, respectively. Based on animal data, the pharmacokinetics of SJ-8029 were predicted in humans using simple allometry and also by several species-invariant time transformations using kallynochron, apolysichron and dienetichron times. The human pharmacokinetic parameters of Cl, V(ss) and t(1/2) predicted by the simple allometry and various species-invariant time methods were 50.4-145.0 l/h, 369.0-579.8 l and 242.0-1448.3 min, respectively. These preliminary parameter values may be useful in designing early pharmacokinetic studies of SJ-8029 in humans.