Defining the RNaseH2 enzyme-initiated ribonucleotide excision repair pathway in Archaea.

Incorporation of ribonucleotides during DNA replication has severe consequences for genome stability. Although eukaryotes possess a number of redundancies for initiating and completing repair of misincorporated ribonucleotides, archaea such as Thermococcus rely only upon RNaseH2 to initiate the pathway. Because Thermococcus DNA polymerases incorporate as many as 1,000 ribonucleotides per genome, RNaseH2 must be efficient at recognizing and nicking at embedded ribonucleotides to ensure genome integrity. Here, we show that ribonucleotides are incorporated by the hyperthermophilic archaeon Thermococcus kodakarensis both in vitro and in vivo and a robust ribonucleotide excision repair pathway is critical to keeping incorporation levels low in wild-type cells. Using pre-steady-state and steady-state kinetics experiments, we also show that archaeal RNaseH2 rapidly cleaves at embedded ribonucleotides (200-450 s-1), but exhibits an ∼1,000-fold slower turnover rate (0.06-0.17 s-1), suggesting a potential role for RNaseH2 in protecting or marking nicked sites for further processing. We found that following RNaseH2 cleavage, the combined activities of polymerase B (PolB), flap endonuclease (Fen1), and DNA ligase are required to complete ribonucleotide processing. PolB formed a ribonucleotide-containing flap by strand displacement synthesis that was cleaved by Fen1, and DNA ligase sealed the nick for complete repair. Our study reveals conservation of the overall mechanism of ribonucleotide excision repair across domains of life. The lack of redundancies in ribonucleotide repair in archaea perhaps suggests a more ancestral form of ribonucleotide excision repair compared with the eukaryotic pathway.

The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis.

Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5'-to-3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase.IMPORTANCE The mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer removal in Archaea Here we demonstrate that in Thermococcus kodakarensis, either Fen1 or GAN activity is sufficient for viability. Furthermore, GAN can support growth in the absence of both Fen1 and RNase HII, but Fen1 and RNase HII are required for viability in the absence of GAN.

Subunit connectivity, assembly determinants and architecture of the yeast exocyst complex.

The exocyst is a hetero-octameric complex that has been proposed to serve as the tethering complex for exocytosis, although it remains poorly understood at the molecular level. Here, we purified endogenous exocyst complexes from Saccharomyces cerevisiae and showed that they are stable and consist of all eight subunits with equal stoichiometry. Using a combination of biochemical and auxin induced-degradation experiments in yeast, we mapped the subunit connectivity, identified two stable four-subunit modules within the octamer and demonstrated that several known exocyst-binding partners are not necessary for exocyst assembly and stability. Furthermore, we visualized the structure of the yeast complex by using negative-stain electron microscopy; our results indicate that the exocyst exists predominantly as a stable, octameric complex with an elongated architecture that suggests that the subunits are contiguous helical bundles packed together into a bundle of long rods.

Exorcising the exocyst complex.

The exocyst complex is an evolutionarily conserved multisubunit protein complex implicated in tethering secretory vesicles to the plasma membrane. Originally identified two decades ago in budding yeast, investigations using several different eukaryotic systems have since made great progress toward determination of the overall structure and organization of the eight exocyst subunits. Studies point to a critical role for the complex as a spatiotemporal regulator through the numerous protein and lipid interactions of its subunits, although a molecular understanding of exocyst function has been challenging to elucidate. Recent progress demonstrates that the exocyst is also important for additional trafficking steps and cellular processes beyond exocytosis, with links to development and disease. In this review, we discuss current knowledge of exocyst architecture, assembly, regulation and its roles in a variety of cellular trafficking pathways.