Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

Promoter Distortion and Opening in the RNA Polymerase II Cleft.

Transcription initiation requires opening of promoter DNA in the RNA polymerase II (Pol II) pre-initiation complex (PIC), but it remains unclear how this is achieved. Here we report the cryo-electron microscopic (cryo-EM) structure of a yeast PIC that contains underwound, distorted promoter DNA in the closed Pol II cleft. The DNA duplex axis is offset at the upstream edge of the initially melted DNA region (IMR) where DNA opening begins. Unstable IMRs are found in a subset of yeast promoters that we show can still initiate transcription after depletion of the transcription factor (TF) IIH (TFIIH) translocase Ssl2 (XPB in human) from the nucleus in vivo. PIC-induced DNA distortions may thus prime the IMR for melting and may explain how unstable IMRs that are predicted in promoters of Pol I and Pol III can open spontaneously. These results suggest that DNA distortion in the polymerase cleft is a general mechanism that contributes to promoter opening.

Exo1 competes with repair synthesis, converts NER intermediates to long ssDNA gaps, and promotes checkpoint activation.

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism may be stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability.

Short nucleosome repeats impose rotational modulations on chromatin fibre folding.

In eukaryotic cells, DNA is organized into arrays of repeated nucleosomes where the shorter nucleosome repeat length (NRL) types are associated with transcriptionally active chromatin. Here, we tested a hypothesis that systematic variations in the NRL influence nucleosome array folding into higher-order structures. For NRLs with fixed rotational settings, we observed a negative correlation between NRL and chromatin folding. Rotational variations within a range of longer NRLs (188 bp and above) typical of repressed chromatin in differentiated cells did not reveal any changes in chromatin folding. In sharp contrast, for the shorter NRL range of 165-177 bp, we observed a strong periodic dependence of chromatin folding upon the changes in linker DNA lengths, with the 172 bp repeat found in highly transcribed yeast chromatin imposing an unfolded state of the chromatin fibre that could be reversed by linker histone. Our results suggest that the NRL may direct chromatin higher-order structure into either a nucleosome position-dependent folding for short NRLs typical of transcribed genes or an architectural factor-dependent folding typical of longer NRLs prevailing in eukaryotic heterochromatin.

Global mapping of DNA conformational flexibility on Saccharomyces cerevisiae.

In this study we provide the first comprehensive map of DNA conformational flexibility in Saccharomyces cerevisiae complete genome. Flexibility plays a key role in DNA supercoiling and DNA/protein binding, regulating DNA transcription, replication or repair. Specific interest in flexibility analysis concerns its relationship with human genome instability. Enrichment in flexible sequences has been detected in unstable regions of human genome defined fragile sites, where genes map and carry frequent deletions and rearrangements in cancer. Flexible sequences have been suggested to be the determinants of fragile gene proneness to breakage; however, their actual role and properties remain elusive. Our in silico analysis carried out genome-wide via the StabFlex algorithm, shows the conserved presence of highly flexible regions in budding yeast genome as well as in genomes of other Saccharomyces sensu stricto species. Flexibile peaks in S. cerevisiae identify 175 ORFs mapping on their 3'UTR, a region affecting mRNA translation, localization and stability. (TA)n repeats of different extension shape the central structure of peaks and co-localize with polyadenylation efficiency element (EE) signals. ORFs with flexible peaks share common features. Transcripts are characterized by decreased half-life: this is considered peculiar of genes involved in regulatory systems with high turnover; consistently, their function affects biological processes such as cell cycle regulation or stress response. Our findings support the functional importance of flexibility peaks, suggesting that the flexible sequence may be derived by an expansion of canonical TAYRTA polyadenylation efficiency element. The flexible (TA)n repeat amplification could be the outcome of an evolutionary neofunctionalization leading to a differential 3'-end processing and expression regulation in genes with peculiar function. Our study provides a new support to the functional role of flexibility in genomes and a strategy for its characterization inside human fragile sites.

Nucleosome positioning by genomic excluding-energy barriers.

Recent genome-wide nucleosome mappings along with bioinformatics studies have confirmed that the DNA sequence plays a more important role in the collective organization of nucleosomes in vivo than previously thought. Yet in living cells, this organization also results from the action of various external factors like DNA-binding proteins and chromatin remodelers. To decipher the code for intrinsic chromatin organization, there is thus a need for in vitro experiments to bridge the gap between computational models of nucleosome sequence preferences and in vivo nucleosome occupancy data. Here we combine atomic force microscopy in liquid and theoretical modeling to demonstrate that a major sequence signaling in vivo are high-energy barriers that locally inhibit nucleosome formation rather than favorable positioning motifs. We show that these genomic excluding-energy barriers condition the collective assembly of neighboring nucleosomes consistently with equilibrium statistical ordering principles. The analysis of two gene promoter regions in Saccharomyces cerevisiae and the human genome indicates that these genomic barriers direct the intrinsic nucleosome occupancy of regulatory sites, thereby contributing to gene expression regulation.

Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy.

The structure of the multisubunit yeast DNA polymerase epsilon (Pol epsilon) was determined to 20-A resolution using cryo-EM and single-particle image analysis. A globular domain comprising the catalytic Pol2 subunit is flexibly connected to an extended structure formed by subunits Dpb2, Dpb3 and Dpb4. Consistent with the reported involvement of the latter in interaction with nucleic acids, the Dpb portion of the structure directly faces a single cleft in the Pol2 subunit that seems wide enough to accommodate double-stranded DNA. Primer-extension experiments reveal that Pol epsilon processivity requires a minimum length of primer-template duplex that corresponds to the dimensions of the extended Dpb structure. Together, these observations suggest a mechanism for interaction of Pol epsilon with DNA that might explain how the structure of the enzyme contributes to its intrinsic processivity.

Role of nucleic acid binding in Sir3p-dependent interactions with chromatin fibers.

Recent studies of the mechanisms involved in the regulation of gene expression in eukaryotic organisms depict a highly complex process requiring a coordinated rearrangement of numerous molecules to mediate DNA accessibility. Silencing in Saccharomyces cerevisiae involves the Sir family of proteins. Sir3p, originally described as repressing key areas of the yeast genome through interactions with the tails of histones H3 and H4, appears to have additional roles in that process, including involvement with a DNA binding component. Our in vitro studies focused on the characterization of Sir3p-nucleic acid interactions and their biological functions in Sir3p-mediated silencing using binding assays, EM imaging, and theoretical modeling. Our results suggest that the initial Sir3p recruitment is partially DNA-driven, highly cooperative, and dependent on nucleosomal features other than histone tails. The initial step appears to be rapidly followed by the spreading of silencing using linker DNA as a track.

Observation of individual DNA molecules undergoing gel electrophoresis.

Individual DNA molecules undergoing agarose gel electrophoresis were viewed with the aid of a fluorescence microscope. Molecular shape and orientation were studied in both steady and pulsed electric fields. It was observed that (i) DNA macromolecules advanced lengthwise through the gel in an extended configuration, (ii) the molecules alternately contracted and lengthened as they moved, (iii) the molecules often became hooked around obstacles in a U-shape for extended periods, and (iv) the molecules displayed elasticity as they extended from both ends at once. A computer model has been developed that simulates the migration of the molecules in a rotating-field gel electrophoresis experiment.

Electron microscopy methods for studying in vivo DNA replication intermediates.

The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen crosslinking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncover the basic mechanisms of DNA duplication, as well as the perturbation of this process by genotoxic treatments. The replication structures need to the stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, finally leading to the visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize in vivo replication intermediates of genomic DNA, extracted from budding yeast or cultured mammalian cells.

On-site manipulation of single chromosomal DNA molecules by using optically driven microstructures.

We report a novel method for manipulation of single giant DNA molecules under a video microscope. Using optically driven microstructures, we manipulated chromosomal DNA of length in the order of millimetres, extended by electroosmotic flow without DNA breakage in aqueous solution: we picked up DNA, using microfabricated hooks and wound it around microfabricated bobbins.

Recombination at long mutant telomeres produces tiny single- and double-stranded telomeric circles.

Recombinational telomere elongation (RTE) known as alternate lengthening of telomeres is the mechanism of telomere maintenance in up to 5 to 10% of human cancers. The telomeres of yeast mutants lacking telomerase can also be maintained by recombination. Previously, we proposed the roll-and-spread model to explain this elongation in the yeast Kluveromyces lactis. This model suggests that a very small ( approximately 100-bp) circular molecule of telomeric DNA is copied by a rolling circle event to generate a single long telomere. The sequence of this primary elongated telomere is then spread by recombination to all remaining telomeres. Here we show by two-dimensional gel analysis and electron microscopy that small circles of single- and double-stranded telomeric DNA are commonly made by recombination in a K. lactis mutant with long telomeres. These circles were found to be especially abundant between 100 and 400 bp (or nucleotides). Interestingly, the single-stranded circles consist of only the G-rich telomeric strand sequence. To our knowledge this is the first report of single-stranded telomeric circles as a product of telomere dysfunction. We propose that the small telomeric circles form through the resolution of an intratelomeric strand invasion which resembles a t-loop. Our data reported here demonstrate that K. lactis can, in at least some circumstances, make telomeric circles of the very small sizes predicted by the roll-and-spread model. The very small circles seen here are both predicted products of telomere rapid deletion, a process observed in both human and yeast cells, and predicted templates for roll-and-spread RTE.

Dynamic Architecture of Eukaryotic DNA Replication Forks In Vivo, Visualized by Electron Microscopy.

The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.

Tetraplex structure of fission yeast telomeric DNA and unfolding of the tetraplex on the interaction with telomeric DNA binding protein Pot1.

To understand the regulation mechanism of fission yeast telomeric DNA, we analysed the structural properties of Gn: d(GnTTAC) (n=2-6) and 4Gn: d(GnTTAC)4 (n=3 and 4), and their interaction with the single-stranded telomeric DNA binding domain of telomere-binding protein Pot1 (Pot1DBD). G4, G5 and G6 formed a parallel tetraplex in contrast with no tetraplex formation by G2 and G3. Also, 4G4 adopted only an antiparallel tetraplex in spite of a mixture of parallel and antiparallel tetraplexes of 4G3. The variety of tetraplex structures was governed by the number of consecutive guanines in a single copy and the number of repeats. The antiparallel tetraplex of 4G4 became unfolded upon the interaction with Pot1DBD. The interaction with mutant Pot1DBD proteins revealed that the ability to unfold the antiparallel tetraplex was strongly correlated with the specific binding affinity for the single-stranded telomeric DNA. The result suggests that the decrease in the free single strand upon the complex formation with Pot1DBD may shift the equilibrium from the tetraplex to the single strand, which may cause the tetraplex unfolding. Considering that the antiparallel tetraplex inhibits telomerase-mediated telomere elongation, we conclude that the ability of Pot1 to unfold the antiparallel tetraplex is required for telomerase-mediated telomere regulation.

Electron microscopy of Achlya deoxyribonucleic acid sequence organization.

Electron microscopic analysis of reassociated deoxyribonucleic acid (DNA) from the aquatic fungus Achlya bisexualis revealed details of the sequence arrangement of the inverted repeats and both the highly and moderately repetitive sequence clusters. We used the gene 32 protein-ethidium bromide technique for visualizing the DNA molecules, a procedure which provides excellent contrast between single- and double-stranded DNA regions. Long (greater than 6-kilobase) DNA fragments were isolated after reannealing to two different repetitive C0t values, and the renatured structures were then visualized in an electron microscope. Our results showed that the inverted repeat sequences were short (0.5 kilobase, number-average) and separated by nonhomologous DNA of various lengths. These pairs of sequences were not clustered within the genome. Both highly repetitive and moderately repetitive DNA sequences were organized as tandem arrays of precisely paired, regularly repeating units. No permuted clusters of repeating sequences were observed, nor was there evidence of interspersion of repetitive with single-copy DNA sequences in the Achlya genome.

Physical detection of heteroduplexes during meiotic recombination in the yeast Saccharomyces cerevisiae.

We describe a general physical method for detecting the heteroduplex DNA that is formed as an intermediate in meiotic recombination in the yeast Saccharomyces cerevisiae. We use this method to study the kinetic relationship between the formation of heteroduplex DNA and other meiotic events. We show that strains with the rad50, but not the rad52, mutation are defective in heteroduplex formation. We also demonstrate that, although cruciform structures can be formed in vivo as a consequence of heteroduplex formation between DNA strands that contain different palindromic insertions, small palindromic sequences in homoduplex DNA are rarely extruded into the cruciform conformation.

Topoisomerase function during replication-independent chromatin assembly in yeast.

DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach. As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2). Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone. Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II. Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I. We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells.

In vivo analysis of the Saccharomyces cerevisiae HO nuclease recognition site by site-directed mutagenesis.

HO nuclease introduces a specific double-strand break in the mating-type locus (MAT) of Saccharomyces cerevisiae, initiating mating-type interconversion. To define the sequence recognized by HO nuclease, random mutations were produced in a 30-base-pair region homologous to either MAT alpha or MATa by a chemical synthesis procedure. The mutant sites were introduced into S. cerevisiae on a shuttle vector and tested for the ability to stimulate recombination in an assay that mimics mating-type interconversion. The results suggest that a core of 8 noncontiguous bases near the Y-Z junction of MAT is essential for HO nuclease to bind and cleave its recognition site. Other contacts must be required because substrates that contain several mutations outside an intact core reduce or eliminate cleavage in vivo. The results show that HO site recognition is a complex phenomenon, similar to promoter-polymerase interactions.

Linear mitochondrial DNAs of yeasts: closed-loop structure of the termini and possible linear-circular conversion mechanisms.

The terminal structure of the linear mitochondrial DNA (mtDNA) from three yeast species has been examined. By enzymatic digestion, alkali denaturation, and sequencing of cloned termini, it was shown that in Pichia pijperi and P. jadinii, both termini of the linear mtDNA were made of a single-stranded loop covalently joining the two strands, as in the case of vaccinia virus DNA. The left and right loop sequences were in either of two orientations, suggesting the existence of a flip-flop inversion mechanism. Contiguous to the terminal loops, inverted terminal repeats were present. The mtDNA from Williopsis mrakii seems to have an analogous structure, although terminal loops could not be directly demonstrated. Electron microscopy revealed the presence, among linear molecules, of a small number of circular DNAs, mostly of monomer length. Linear and circular models of replication are considered, and possible conversion mechanisms between linear and circular forms are discussed. A flip-flop inversion mechanism between the inverted repeat sequences within a circular intermediate may be involved in the generation of the linear form of mtDNA.

Transcription in the yeast rRNA gene locus: distribution of the active gene copies and chromatin structure of their flanking regulatory sequences.

In growing yeast cells, about half of the 150 tandemly repeated rRNA genes are transcriptionally active and devoid of nucleosomes. By using the intercalating drug psoralen as a tool to mark accessible sites along chromatin DNA in vivo, we found that the active rRNA gene copies are rather randomly distributed along the ribosomal rRNA gene locus. Moreover, results from the analysis of a single, tagged transcription unit in the tandem array are not consistent with the presence of a specific subset of active genes that is stably maintained throughout cell divisions. In the rRNA intergenic spacers of yeast cells, an enhancer is located at the 3' end of each transcription unit, 2 kb upstream of the next promoter. Analysis of the chromatin structure along the tandem array revealed a structural link between transcription units and adjacent, 3' flanking enhancer sequences: each transcriptionally active gene is flanked by a nonnucleosomal enhancer, whereas inactive, nucleosome-packed gene copies are followed by enhancers regularly packaged in nucleosomes. From the fact that nucleosome-free enhancers were also detected in an RNA polymerase I mutant strain, we interpret these open chromatin structures as being the result of specific protein-DNA interactions that can occur before the onset of transcription. In contrast, in this mutant strain, all of the rRNA coding sequences are packaged in nucleosomal arrays. This finding indicates that the establishment of the open chromatin conformation on the activated gene copies requires elongating RNA polymerase I molecules advancing through the template.