An exogenous chloroplast genome for complex sequence manipulation in algae.

We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.

DNA polymerase epsilon, acetylases and remodellers cooperate to form a specialized chromatin structure at a tRNA insulator.

Insulators bind transcription factors and use chromatin remodellers and modifiers to mediate insulation. In this report, we identified proteins required for the efficient formation and maintenance of a specialized chromatin structure at the yeast tRNA insulator. The histone acetylases, SAS-I and NuA4, functioned in insulation, independently of tRNA and did not participate in the formation of the hypersensitive site at the tRNA. In contrast, DNA polymerase epsilon, functioned with the chromatin remodeller, Rsc, and the histone acetylase, Rtt109, to generate a histone-depleted region at the tRNA insulator. Rsc and Rtt109 were required for efficient binding of TFIIIB to the tRNA insulator, and the bound transcription factor and Rtt109 in turn were required for the binding of Rsc to tRNA. Robust insulation during growth and cell division involves the formation of a hypersensitive site at the insulator during chromatin maturation together with competition between acetylases and deacetylases.

H2A.Z functions to regulate progression through the cell cycle.

Histone H2A variants are highly conserved proteins found ubiquitously in nature and thought to perform specialized functions in the cell. Studies in yeast on the histone H2A variant H2A.Z have shown a role for this protein in transcription as well as chromosome segregation. Our studies have focused on understanding the role of H2A.Z during cell cycle progression. We found that htz1delta cells were delayed in DNA replication and progression through the cell cycle. Furthermore, cells lacking H2A.Z required the S-phase checkpoint pathway for survival. We also found that H2A.Z localized to the promoters of cyclin genes, and cells lacking H2A.Z were delayed in the induction of these cyclin genes. Several different models are proposed to explain these observations.

Rad53 regulates replication fork restart after DNA damage in Saccharomyces cerevisiae.

Replication fork stalling at a DNA lesion generates a damage signal that activates the Rad53 kinase, which plays a vital role in survival by stabilizing stalled replication forks. However, evidence that Rad53 directly modulates the activity of replication forks has been lacking, and the nature of fork stabilization has remained unclear. Recently, cells lacking the Psy2-Pph3 phosphatase were shown to be defective in dephosphorylation of Rad53 as well as replication fork restart after DNA damage, suggesting a mechanistic link between Rad53 deactivation and fork restart. To test this possibility we examined the progression of replication forks in methyl-methanesulfonate (MMS)-damaged cells, under different conditions of Rad53 activity. Hyperactivity of Rad53 in pph3Delta cells slows fork progression in MMS, whereas deactivation of Rad53, through expression of dominant-negative Rad53-KD, is sufficient to allow fork restart during recovery. Furthermore, combined deletion of PPH3 and PTC2, a second, unrelated Rad53 phosphatase, results in complete replication fork arrest and lethality in MMS, demonstrating that Rad53 deactivation is a key mechanism controlling fork restart. We propose a model for regulation of replication fork progression through damaged DNA involving a cycle of Rad53 activation and deactivation that coordinates replication restart with DNA repair.

Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage.

Activation of the checkpoint kinase Rad53 is a critical response to DNA damage that results in stabilization of stalled replication forks, inhibition of late-origin initiation, up-regulation of dNTP levels, and delayed entry to mitosis. Activation of Rad53 is well understood and involves phosphorylation by the protein kinases Mec1 and Tel1 as well as in trans autophosphorylation by Rad53 itself. However, deactivation of Rad53, which must occur to allow the cell to recover from checkpoint arrest, is not well understood. Here, we present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3-Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate-mediated DNA damage. In the absence of Pph3-Psy2, Rad53 dephosphorylation and the resumption of DNA synthesis are delayed during recovery from DNA damage. This delay in DNA synthesis reflects a failure to restart stalled replication forks, whereas, remarkably, genome replication is eventually completed by initiating late origins of replication despite the presence of hyperphosphorylated Rad53. These findings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.

Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae.

Mrc1 associates with replication forks, where it transmits replication stress signals and is required for normal replisome pausing in response to nucleotide depletion. Mrc1 also plays a poorly understood role in DNA replication, which appears distinct from its role in checkpoint signaling. Here, we demonstrate that Mrc1 functions constitutively to promote normal replication fork progression. In mrc1Delta cells, replication forks proceed slowly throughout chromatin, rather than being specifically defective in pausing and progression through loci that impede fork progression. Analysis of genetic interactions with Rrm3, a DNA helicase required to resolve paused forks, indicates that Mrc1 checkpoint signaling is dispensable for the resolution of stalled replication forks and suggests that replication forks lacking Mrc1 create DNA damage that must be repaired by Rrm3. These findings elucidate a central role for Mrc1 in normal replisome function, which is distinct from its role as a checkpoint mediator, but nevertheless critical to genome stability.