An 8-fold βα barrel protein with redundant folding possibilities

Protein sequences containing redundant segments of secondary structure at both termini have the choice a priori of folding into several possible circularly permuted variants of the wild-type tertiary structure. To test this hypothesis the gene of phosphoribosyl anthranilate isomerase from yeast, which is a single-domain 8-fold βα barrel protein, was modified to produce a 10-fold βα homologue in Escherichia coli. It contained a duplicate of the two C-terminal βα units of supersecondary structure fused to its N-terminus. Most of the protein was recovered from the insoluble fraction of disrupted cells by dissolution in guanidinium chloride solutions and refolding. Pristine protein was purified from the soluble fraction. The purified (βα)10 proteins were enzymically almost fully active. Absorbance, fluorescence and circular dichroism spectra as well as the reversible unfolding behaviour of both proteins were also very similar to the properties of the original (βα)8 protein. Digestion with endopeptidases converted both the pristine and the refolded (βα)10 variant to the same large fragment that had the N-terminal sequence and mol. wt of the wild-type βα)8 protein. The data suggest that the folding of the (βα)10 variant is controlled thermodynamically both in vivo and in vitr

Histone Methylation: Navigating the structure of COMPASS

Cryo-electron microscopy reveals how ubiquitination promotes the methylation of histone H3 by the histone-modifying complex COMPASS. </div

Archaeal chromatin 'slinkies' are inherently dynamic complexes with deflected DNA wrapping pathways

Eukaryotes and many archaea package their DNA with histones. While the four eukaryotic histones wrap ~147 DNA base pairs into nucleosomes, archaeal histones form &lsquo;nucleosome-like&rsquo; complexes that continuously wind between 60 and 500 base pairs of DNA (&lsquo;archaeasomes&rsquo;), suggested by crystal contacts and analysis of cellular chromatin. Solution structures of large archaeasomes (&gt;90 DNA base pairs) have never been directly observed. Here, we utilize molecular dynamics simulations, analytical ultracentrifugation, and cryoEM to structurally characterize the solution state of archaeasomes on longer DNA. Simulations reveal dynamics of increased accessibility without disruption of DNA-binding or tetramerization interfaces. Mg2+ concentration influences compaction, and cryoEM densities illustrate that DNA is wrapped in consecutive substates arranged 90o out-of-plane with one another. Without ATP-dependent remodelers, archaea may leverage these inherent dynamics to balance chromatin packing and accessibility. </div

Poly(ADP-ribose) polymerase 1 searches DNA via a 'monkey bar' mechanism.

Poly(ADP-ribose) polymerase 1 (PARP1) is both a first responder to DNA damage and a chromatin architectural protein. How PARP1 rapidly finds DNA damage sites in the context of a nucleus filled with undamaged DNA, to which it also binds, is an unresolved question. Here, we show that PARP1 association with DNA is diffusion-limited, and release of PARP1 from DNA is promoted by binding of an additional DNA molecule that facilitates a 'monkey bar' mechanism, also known as intersegment transfer. The WGR-domain of PARP1 is essential to this mechanism, and a point mutation (W589A) recapitulates the altered kinetics of the domain deletion. Demonstrating the physiological importance of the monkey bar mechanism for PARP1 function, the W589A mutant accumulates at sites of DNA damage more slowly following laser micro-irradiation than wild-type PARP1. Clinically relevant inhibitors of PARP1 did not alter the rate or mechanism of the release of PARP1 from DNA

Solution structure(s) of trinucleosomes from contrast variation SAXS

Nucleosomes in all eukaryotic cells are organized into higher order structures that facilitate genome compaction. Visualizing these organized structures is an important step in understanding how genomic DNA is efficiently stored yet remains accessible to information-processing machinery. Arrays of linked nucleosomes serve as useful models for understanding how the properties of both DNA and protein partners affect their arrangement. A number of important questions are also associated with understanding how the spacings between nucleosomes are affected by the histone proteins, chromatin remodelers, or other chromatin-associated protein partners. Contrast variation small angle X-ray scattering (CVSAXS) reports the DNA conformation within protein-DNA complexes and here is applied to measure the conformation(s) of trinucleosomes in solution, with specific sensitivity to the distance between and relative orientation of linked nucleosomes. These data are interpreted in conjunction with DNA models that account for its sequence dependent mechanical properties, and Monte-Carlo techniques that generate realistic structures for comparison with measured scattering profiles. In solution, trinucleosomes segregate into two dominant populations, with the flanking nucleosomes stacked or nearly equilaterally separated, e.g. with roughly equal distance between all pairs of nucleosomes. These populations are consistent with previously observed magnesium-dependent structures of trinucleosomes with shorter linkers. &nbsp;</p

HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase

Poly(ADP-ribose) polymerase 1 (PARP1) is an important player in the response to DNA damage. Recently, Histone PARylation Factor (HPF1) was shown to be a critical modulator of the activity of PARP1 by facilitating PARylation of histones and redirecting the target amino acid specificity from acidic to serine residues. Here, we investigate the mechanism and specific consequences of HPF1-mediated PARylation using nucleosomes as both activators and substrates for PARP1. HPF1 provides that catalytic base Glu284 to substantially redirect PARylation by PARP1 such that the histones in nucleosomes become the primary recipients of PAR chains. Surprisingly, HPF1 partitions most of the reaction product to free ADP-ribose (ADPR), resulting in much shorter PAR chains compared to reactions in the absence of HPF1. This HPF1-mediated switch from polymerase to hydrolase has important implications for the PARP1-mediated response to DNA damage and raises interesting new questions about the role of intracellular ADPR and depletion of NAD+. </div

PARP inhibitors trap PARP2 and alter the mode of recruitment of PARP2 at DNA damage sites

Dual-inhibitors of PARP1 and PARP2 are promising anti-cancer drugs. In addition to blocking PARP1&amp;2 enzymatic activity, PARP inhibitors also extend the lifetime of DNA damage-induced PARP1&amp;2 foci, termed trapping. Trapping is important for the therapeutic effects of PARP inhibitors. Using live-cell imaging, we found that PARP inhibitors cause persistent PARP2 foci by switching the mode of PARP2 recruitment from a predominantly PARP1- and PAR-dependent rapid exchange to a WGR domain-mediated stalling of PARP2 on DNA. Specifically, PARP1-deletion markedly reduces but does not abolish PARP2 foci. The residual PARP2 foci in PARP1-deficient cells are DNA-dependent and abrogated by the R140A mutation in the WGR domain. Yet, PARP2-R140A forms normal foci in PARP1-proficient cells. In PARP1-deficient cells, PARP inhibitors - niraparib, talazoparib, and, to a lesser extent, olaparib - enhance PARP2 foci by preventing PARP2 exchange. This trapping of PARP2 is independent of auto-PARylation and is abolished by the R140A mutation in the WGR domain and the H415A mutation in the catalytic domain. Taken together, we found that PARP inhibitors trap PARP2 by physically stalling PARP2 on DNA via the WGR-DNA interaction while suppressing the PARP1- and PAR-dependent rapid exchange of PARP2. &nbsp;</p

The Histone Variant H2A.W Defines Heterochromatin and Promotes Chromatin Condensation in Arabidopsis

SummaryHistone variants play crucial roles in gene expression, genome integrity, and chromosome segregation. We report that the four H2A variants in Arabidopsis define different genomic features, contributing to overall genomic organization. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. Similarities in conserved motifs between H2A.W and another H2A variant in metazoans suggest that plants and animals share common mechanisms for heterochromatin condensation

The elongation factor Spn1 is a multi-functional chromatin binding protein.

The process of transcriptional elongation by RNA polymerase II (RNAPII) in a chromatin context involves a large number of crucial factors. Spn1 is a highly conserved protein encoded by an essential gene and is known to interact with RNAPII and the histone chaperone Spt6. Spn1 negatively regulates the ability of Spt6 to interact with nucleosomes, but the chromatin binding properties of Spn1 are largely unknown. Here, we demonstrate that full length Spn1 (amino acids 1-410) binds DNA, histones H3-H4, mononucleosomes and nucleosomal arrays, and has weak nucleosome assembly activity. The core domain of Spn1 (amino acids 141-305), which is necessary and sufficient in Saccharomyces cerevisiae for growth under ideal growth conditions, is unable to optimally interact with histones, nucleosomes and/or DNA and fails to assemble nucleosomes in vitro. Although competent for binding with Spt6 and RNAPII, the core domain derivative is not stably recruited to the CYC1 promoter, indicating chromatin interactions are an important aspect of normal Spn1 functions in vivo. Moreover, strong synthetic genetic interactions are observed with Spn1 mutants and deletions of histone chaperone genes. Taken together, these results indicate that Spn1 is a histone binding factor with histone chaperone functions