Concerted and differential actions of two enzymatic domains underlie Rad5 contributions to DNA damage tolerance

Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5′s helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.     

Butyric acid production from red algae by a newly isolated <Emphasis Type="Italic">Clostridium</Emphasis> sp. S1

Objective

To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required.

Results

A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5–2 g levulinic acid/l and recovered from HMF inhibition at 0.6–2.5 g/l, resulting in 85–92 % butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l.

Conclusion

Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

     

Highly reproducible quantification of apoptotic cells using micropatterned culture of neurons

The quantification of apoptotic cells is an integral component of many cell-based assays in biological studies. However, current methods for quantifying apoptotic cells using conventional random cultures have shown great limitations, especially for the quantification of primary neurons. Randomly distributed neurons under primary culture conditions can lead to biased estimates, and vastly different estimates of cell numbers can be produced within the same experiment. In this study, we developed a simple, accurate, and reliable technique for quantifying apoptotic neurons by means of micropatterned cell cultures. A polydimethylsiloxane (PDMS) microstencil was used as a physical mask for micropatterning cell cultures, and primary granular neurons (GNs) were successfully cultured within the micropattern-confined regions and homogeneously distributed over the entire field of each pattern. As compared with the conventional method based on random cultures, the micropatterned culture method allowed for highly reproducible quantification of apoptotic cells. These results were also confirmed by using GNs derived from mice with neurodegeneration. We hope that this micropatterning method based on the use of a PDMS microstencil can overcome the technical obstacles existing in current biological studies and will serve as a powerful tool for facilitating the study of apoptosis-involved diseases.