DNA replication and the GINS complex: localization on extended chromatin fibers

<p>Abstract</p> <p>Background</p> <p>The GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex. Here, we show the organization of GINS complexes on extended chromatin fibers in relation to sites of DNA replication and replication-associated proteins.</p> <p>Results</p> <p>Using immunofluorescence microscopy we were able to visualize ORC1, ORC2, PCNA, and GINS complex proteins Psf1 and Psf2 bound to extended chromatin fibers. We were also able to detect these proteins concurrently with the visualization of tracks of recently replicated DNA where EdU, a thymidine analog, was incorporated. This allowed us to assess the chromatin association of proteins of interest in relation to the process of DNA replication. ORC and GINS proteins were found on chromatin fibers before replication could be detected. These proteins were also associated with newly replicated DNA in bead-like structures. Additionally, GINS proteins co-localized with PCNA at sites of active replication.</p> <p>Conclusion</p> <p>In agreement with its proposed role in the initiation of DNA replication, GINS proteins associated with chromatin near sites of ORC binding that were devoid of EdU (absence of DNA replication). The association of GINS proteins with PCNA was consistent with a role in the process of elongation. Additionally, the large size of our chromatin fibers (up to approximately 7 Mb) allowed for a more expansive analysis of the distance between active replicons than previously reported.</p

The Set2/Rpd3S Pathway Suppresses Cryptic Transcription without Regard to Gene Length or Transcription Frequency

In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. It was previously reported that this “cryptic” transcription occurs preferentially in long genes, and in genes that are infrequently transcribed. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. We find that suppression of cryptic transcription occurs independent of gene length or transcriptional frequency. Our conclusions differ with those reported previously because we obtained a higher-resolution dataset, we accounted for the fact that gene length and transcriptional frequency are not independent variables, and we accounted for several ascertainment biases that make cryptic transcription easier to detect in long, infrequently transcribed genes. These new results and conclusions have implications for many commonly used genomic analysis approaches, and for the evolution of high-fidelity RNA polymerase II transcriptional initiation in eukaryotes

Sub-Telomeric core X and Y' Elements in S.cerevisiae Suppress Extreme Variations in Gene Silencing

Telomere Position Effect (TPE) is governed by strong repression signals emitted by telomeres via the Sir2/3/4 Histone Deacetylase complex. These signals are then relayed by weak proto-silencers residing in the subtelomeric core X and Y' elements. Subtelomeres also contain Sub-Telomeric Anti-silencing Regions (STARs). In this study we have prepared telomeres built of different combinations of core X, Y' and STARs and have analyzed them in strains lacking Histone-Acetyltransferase genes as well as in cdc6-1 and Δrif1 strains. We show that core X and Y' dramatically reduce both positive and negative variations in TPE, that are caused by these mutations. We also show that the deletion of Histone-Acetyltransferase genes reduce the silencing activity of an ACS proto-silencer, but also reduce the anti-silencing activity of a STAR. We postulate that core X and Y' act as epigenetic “cushioning” cis-elements

Mutation of the Zebrafish Nucleoporin elys Sensitizes Tissue Progenitors to Replication Stress

The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress

Contributions of Histone H3 Nucleosome Core Surface Mutations to Chromatin Structures, Silencing and DNA Repair

Histone H3 mutations in residues that cluster in a discrete region on the nucleosome surface around lysine 79 of H3 affect H3-K79 methylation, impair transcriptional silencing in subtelomeric chromatin, and reveal distinct contributions of histone H3 to various DNA-damage response and repair pathways. These residues might act by recruitment of silencing and DNA-damage response factors. Alternatively, their location on the nucleosome surface suggests a possible involvement in nucleosome positioning, stability and nucleosome interactions. Here, we show that the yeast H3 mutants hht2-T80A, hht2-K79E, hht2-L70S, and hht2-E73D show normal nucleosome positioning and stability in minichromosomes. However, loss of silencing in a subtelomeric URA3 gene correlates with a shift of the promoter nucleosome, while nucleosome positions and stability in the coding region are maintained. Moreover, the H3 mutants show normal repair of UV lesions by photolyase and nucleotide excision repair in minichromosomes and slightly enhanced repair in the subtelomeric region. Thus, these results support a role of those residues in the recruitment of silencing proteins and argue against a general role in nucleosome organization

Histone H4 Lysine 12 Acetylation Regulates Telomeric Heterochromatin Plasticity in Saccharomyces cerevisiae

Recent studies have established that the highly condensed and transcriptionally silent heterochromatic domains in budding yeast are virtually dynamic structures. The underlying mechanisms for heterochromatin dynamics, however, remain obscure. In this study, we show that histones are dynamically acetylated on H4K12 at telomeric heterochromatin, and this acetylation regulates several of the dynamic telomere properties. Using a de novo heterochromatin formation assay, we surprisingly found that acetylated H4K12 survived the formation of telomeric heterochromatin. Consistently, the histone acetyltransferase complex NuA4 bound to silenced telomeric regions and acetylated H4K12. H4K12 acetylation prevented the over-accumulation of Sir proteins at telomeric heterochromatin and elimination of this acetylation caused defects in multiple telomere-related processes, including transcription, telomere replication, and recombination. Together, these data shed light on a potential histone acetylation mark within telomeric heterochromatin that contributes to telomere plasticity

Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing

We want to thank Kathy Kyler for editing this manuscript, Ken Stuart for supplying monoclonal antisera against RECC subunits, and Laurie K. Read for her gift of polyclonal antisera against GAP1 and RGG2. Funding: National Science Foundation Grant No. NSF1122109 (PI: J.Cruz-Reyes.). NIH/National Institute of Allergies and Infectious Diseases R01 AI088011 (PI: Blaine Mooers). Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103640. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.Yeshttp://www.plosone.org/static/editorial#pee