Nucleosomes influence multiple steps during replication initiation

Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.American Cancer Society (Postdoctoral Fellowship 123700-PF-13-071-01)National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051

Diversity of Eukaryotic DNA Replication Origins Revealed by Genome-Wide Analysis of Chromatin Structure

Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function

Lsr2 of Mycobacterium tuberculosis is a DNA-bridging protein

Lsr2 is a small, basic protein present in Mycobacterium and related actinomycetes. Recent studies suggest that Lsr2 is a regulatory protein involved in multiple cellular processes including cell wall biosynthesis and antibiotic resistance. However, the underlying molecular mechanisms remain unknown. In this article, we performed biochemical studies of Lsr2–DNA interactions and structure–function analysis of Lsr2. Analysis by atomic force microscopy revealed that Lsr2 has the ability to bridge distant DNA segments, suggesting that Lsr2 plays a role in the overall organization and compactness of the nucleoid. Mutational analysis identified critical residues and selection of dominant negative mutants demonstrated that both DNA binding and protein oligomerization are essential for the normal functions of Lsr2 in vivo. These results provide strong evidence that Lsr2 is a DNA bridging protein, which represents the first identification of such proteins in bacteria phylogenetically distant from the Enterobacteriaceae. DNA bridging by Lsr2 also provides a mechanism of transcriptional regulation by Lsr2

The chromatin remodelling enzymes SNF2H and SNF2L position nucleosomes adjacent to CTCF and other transcription

Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase's most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements

Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone

Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian intitiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites

Chromosome Duplication in <i>Saccharomyces cerevisiae</i>

The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G 1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation. Keywords: DNA replication; cell cycle; chromatin; chromosome duplication; genome stability; YeastBookNational Institutes of Health (U.S.) (Grant GM-052339

Genome-wide Analysis of Nucleosome Occupancy Surrounding Saccharomyces cerevisiae Origins of Replication

The Saccharomyces cerevisiae origin recognition complex (ORC) binds to replication origins at the ARS consensus sequence (ACS), serving as a scaffold for the assembly of replication complexes needed for the initiation of DNA synthesis. I generated a genome-wide map of nucleosome positions surrounding replication origins because the precise locations of nucleosomes may influence replication. My map revealed a nucleosome-free region surrounding the ACS that is bordered by two well-positioned nucleosomes. I was able to explain differences in origin properties by clustering nucleosome profiles. I found an association between the replication time and nucleosome profile for a given origin cluster. An ORC depletion mutant nucleosome map indicated a shift in nucleosomes towards the ACS. I present the first genome-wide view of origin nucleosome architecture, indicate a relationship between chromatin structure and replication timing, and suggest a model whereby the interplay between DNA sequence and ORC binding defines the nucleosome occupancy pattern.MAS

Synthesis and structure of [Sb7Ni3(CO)3]3-: a new structural type for nido 10-vertex polyhedral clusters

Forest plot of cardiovascular death in HF with MRA use by MRA type. (EPS 2561 kb