The Amazing Acrobat: Yeast's Histone H3K56 Juggles Several Important Roles While Maintaining Perfect Balance.

Acetylation on lysine 56 of histone H3 of the yeast Saccharomyces cerevisiae has been implicated in many cellular processes that affect genome stability. Despite being the object of much research, the complete scope of the roles played by K56 acetylation is not fully understood even today. The acetylation is put in place at the S-phase of the cell cycle, in order to flag newly synthesized histones that are incorporated during DNA replication. The signal is removed by two redundant deacetylases, Hst3 and Hst4, at the entry to G2/M phase. Its crucial location, at the entry and exit points of the DNA into and out of the nucleosome, makes this a central modification, and dictates that if acetylation and deacetylation are not well concerted and executed in a timely fashion, severe genomic instability arises. In this review, we explore the wealth of information available on the many roles played by H3K56 acetylation and the deacetylases Hst3 and Hst4 in DNA replication and repair.

Histones on fire: the effect of Dun1 and Mrc1 on origin firing and replication of hyper-acetylated genomes.

As cells replicate their DNA, there is a need to synthesize new histones with which to wrap it. Newly synthesized H3 histones that are incorporated into the assembling chromatin behind the replication fork are acetylated at lysine 56. The acetylation is removed by two deacetylases, Hst3 and Hst4. This process is tightly regulated and any perturbation leads to genomic instability and replicative stress. We recently showed that Dun1, a kinase implicated mainly in the regulation of dNTPs, is vital in cells with hyper-acetylation, to counteract Rad53's inhibition on late-firing origins of replication. Our work showed that ∆hst3 ∆hst4 cells depend on late origin firing for survival, and are unable to prevent Rad53's inhibition when Dun1 is inactive. Thus, our work describes a role for Dun1 that is independent on its known function as a regulator of dNTP levels. Here we show that Mrc1 (Claspin in mammals), a protein that moves with the replicating fork and participates in both replication and checkpoint functions, plays also an essential role in the absence of H3K56Ac deacetylation. The sum of the results shown here and in our recent publication suggests that dormant origins are also utilized in these cells, making Mrc1, which regulates firing from these origins, also essential when histone H3 is hyper-acetylated. Thus, cells suffering from hyper-acetylation of H3K56 experience replication stress caused by a combination of prone-to-collapse forks and limited replication tracts. This combination makes both Dun1 and Mrc1, each acting on different targets, essential for viability.

A novel role for Dun1 in the regulation of origin firing upon hyper-acetylation of H3K56.

During DNA replication newly synthesized histones are incorporated into the chromatin of the replicating sister chromatids. In the yeast Saccharomyces cerevisiae new histone H3 molecules are acetylated at lysine 56. This modification is carefully regulated during the cell cycle, and any disruption of this process is a source of genomic instability. Here we show that the protein kinase Dun1 is necessary in order to maintain viability in the absence of the histone deacetylases Hst3 and Hst4, which remove the acetyl moiety from histone H3. This lethality is not due to the well-characterized role of Dun1 in upregulating dNTPs, but rather because Dun1 is needed in order to counteract the checkpoint kinase Rad53 (human CHK2) that represses the activity of late firing origins. Deletion of CTF18, encoding the large subunit of an alternative RFC-like complex (RLC), but not of components of the Elg1 or Rad24 RLCs, is enough to overcome the dependency of cells with hyper-acetylated histones on Dun1. We show that the detrimental function of Ctf18 depends on its interaction with the leading strand polymerase, Polε. Our results thus show that the main problem of cells with hyper-acetylated histones is the regulation of their temporal and replication programs, and uncover novel functions for the Dun1 protein kinase and the Ctf18 clamp loader.

Telomeres and stress in yeast cells: When genes and environment interact.

Telomeres are structures composed of simple DNA repeats and specific proteins that protect the eukaryotic chromosomal ends from degradation, and facilitate the replication of the genome. They are central to the maintenance of the genome integrity, and play important roles in the development of cancer and in the process of aging in humans. The yeast Saccharomyces cerevisiae has greatly contributed to our understanding of basic telomere biology. Our laboratory has carried out systematic screen for mutants that affect telomere length, and identified ∼500 genes that, when mutated, affect telomere length. Remarkably, all ∼500 TLM (Telomere Length Maintenance) genes participate in a very tight homeostatic process, and it is enough to mutate one of them to change the steady-state telomere length. Despite this complex network of balances, it is also possible to change telomere length in yeast by applying several types of external stresses. We summarize our insights about the molecular mechanisms by which genes and environment interact to affect telomere length.

The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51.

Homologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. The Saccharomyces cerevisiae yeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2's helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a new SRS2 allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of the srs2(1-850) allele suggest a role for Srs2's helicase activity in sister chromatid cohesion. Our results also indicate that Srs2's function becomes more central in diploid cells.IMPORTANCE Homologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.