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The accuracy of most DNA processes depends on chromatin integrity and dynamics. Our analyses in the yeast Saccharomyces cerevisiae show that an absence of Swr1 (the catalytic and scaffold subunit of the chromatin-remodeling complex SWR) leads to the formation of long-duration Rad52, but not RPA, foci and to an increase in intramolecular recombination. These phenotypes are further increased by MMS, zeocin, and ionizing radiation, but not by double-strand breaks, HU, or transcription/replication collisions, suggesting that they are associated with specific DNA lesions. Importantly, these phenotypes can be specifically suppressed by mutations in: (1) chromatin-anchorage internal nuclear membrane components (mps3D75-150 and src1D); (2) actin and actin regulators (act1-157, act1-159, crn1D, and cdc42-6); or (3) the SWR subunit Swc5 and the SWR substrate Htz1. However, they are not suppressed by global disruption of actin filaments or by the absence of Csm4 (a component of the external nuclear membrane that forms a bridging complex with Mps3, thus connecting the actin cytoskeleton with chromatin). Moreover, swr1D-induced Rad52 foci and intramolecular recombination are not associated with tethering recombinogenic DNA lesions to the nuclear periphery. In conclusion, the absence of Swr1 impairs efficient recombinational repair of specific DNA lesions by mechanisms that are influenced by SWR subunits, including actin, and nuclear envelope components. We suggest that these recombinational phenotypes might be associated with a pathological effect on homologous recombination of actin-containing complexes. [ABSTRACT FROM AUTHOR]

Transcriptional elongation requires the concerted action of several factors that allow RNA polymerase II to advance through chromatin in a highly processive manner. In order to identify novel elongation factors, we performed systematic yeast genetic screening based on the GLAM (Gene Length-dependent Accumulation of mRNA) assay, which is used to detect defects in the expression of long transcription units. Apart from well-known transcription elongation factors, we identified mutants in the prefoldin complex subunits, which were among those that caused the most dramatic phenotype. We found that prefoldin, so far involved in the cytoplasmic co-translational assembly of protein complexes, is also present in the nucleus and that a subset of its subunits are recruited to chromatin in a transcription-dependent manner. Prefoldin influences RNA polymerase II the elongation rate in vivo and plays an especially important role in the transcription elongation of long genes and those whose promoter regions contain a canonical TATA box. Finally, we found a specific functional link between prefoldin and histone dynamics after nucleosome remodeling, which is consistent with the extensive network of genetic interactions between this factor and the machinery regulating chromatin function. This study establishes the involvement of prefoldin in transcription elongation, and supports a role for this complex in cotranscriptional histone eviction. [ABSTRACT FROM AUTHOR]

The SWR1 complex replaces the canonical histone H2A with the variant H2A.Z (Htz1 in yeast) at specific chromatin regions. This dynamic alteration in nucleosome structure provides a molecular mechanism to regulate transcription, gene silencing, chromosome segregation and DNA repair. Here we show that genetic instability, sensitivity to drugs impairing different cellular processes and genome-wide transcriptional misregulation in htz1Δ can be partially or totally suppressed if SWR1 is not formed (swr1Δ), if it forms but cannot bind to chromatin (swc2Δ) or if it binds to chromatin but lacks histone replacement activity (swc5Δ and the ATPase-dead swr1-K727G). These results suggest that in htz1Δ the nucleosome remodelling activity of SWR1 affects chromatin integrity because of an attempt to replace H2A with Htz1 in the absence of the latter. This would impair transcription and, either directly or indirectly, other cellular processes. Specifically, we show that in htz1Δ, the SWR1 complex causes an accumulation of recombinogenic DNA damage by a mechanism dependent on phosphorylation of H2A at Ser129, a modification that occurs in response to DNA damage, suggesting that the SWR1 complex impairs the repair of spontaneous DNA damage in htz1Δ. In addition, SWR1 causes DSBs sensitivity in htz1Δ; consistently, in the absence of Htz1 the SWR1 complex bound near an endonuclease HO-induced DSB at the mating-type (MAT) locus impairs DSB-induced checkpoint activation. Our results support a stepwise mechanism for the replacement of H2A with Htz1 and demonstrate that a tight control of this mechanism is essential to regulate chromatin dynamics but also to prevent the deleterious consequences of an incomplete nucleosome remodelling. [ABSTRACT FROM AUTHOR]

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, downregulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA-damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication. [ABSTRACT FROM AUTHOR]

Among the collection of chromatin modifications that influence its function and structure, the substitution of canonical histones by the so-called histone variants is one of the most prominent actions. Since crucial meiotic transactions are modulated by chromatin, here we investigate the functional contribution of the H2A.Z histone variant during both unperturbed meiosis and upon challenging conditions where the meiotic recombination checkpoint is triggered in budding yeast by the absence of the synaptonemal complex component Zip1. We have found that H2A.Z localizes to meiotic chromosomes in an SWR1-dependent manner. Although meiotic recombination is not substantially altered, the htz1 mutant (lacking H2A.Z) shows inefficient meiotic progression, impaired sporulation, and reduced spore viability. These phenotypes are likely accounted for by the misregulation of meiotic gene expression landscape observed in htz1. In the zip1 mutant, the absence of H2A.Z results in a tighter meiotic arrest imposed by the meiotic recombination checkpoint. We have found that Mec1-dependent Hop1-T318 phosphorylation and the ensuing Mek1 activation are not significantly altered in zip1 htz1; however, downstream checkpoint targets, such as the meiosis I-promoting factors Ndt80, Cdc5, and Clb1, are drastically downregulated. The study of the checkpoint response in zip1 htz1 has also allowed us to reveal the existence of an additional function of the Swe1 kinase, independent of CDK inhibitory phosphorylation, which is relevant to restrain meiotic cell cycle progression. In summary, our study shows that the H2A.Z histone variant impacts various aspects of meiotic development adding further insight into the relevance of chromatin dynamics for accurate gametogenesis. [ABSTRACT FROM AUTHOR]