Post-translational histone modification has a fundamental role in chromatin biology and is proposed to constitute a 'histone code' in epigenetic regulation. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The discovery of lysyl demethylases using flavin (amine oxidases) or Fe(II) and 2-oxoglutarate as cofactors (2OG oxygenases) has changed the view of methylation as a stable epigenetic marker. However, little is known about how the demethylases are selective for particular lysyl-containing sequences in specific methylation states, a key to understanding their functions. Here we reveal how human JMJD2A (jumonji domain containing 2A), which is selective towards tri- and dimethylated histone H3 lysyl residues 9 and 36 (H3K9me3/me2 and H3K36me3/me2), discriminates between methylation states and achieves sequence selectivity for H3K9. We report structures of JMJD2A-Ni(II)-Zn(II) inhibitor complexes bound to tri-, di- and monomethyl forms of H3K9 and the trimethyl form of H3K36. The structures reveal a lysyl-binding pocket in which substrates are bound in distinct bent conformations involving the Zn-binding site. We propose a mechanism for achieving methylation state selectivity involving the orientation of the substrate methyl groups towards a ferryl intermediate. The results suggest distinct recognition mechanisms in different demethylase subfamilies and provide a starting point to develop chemical tools for drug discovery and to study and dissect the complexity of reversible histone methylation and its role in chromatin biology.

SummaryThe cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.

Producing purified human proteins with high yield and purity remains a considerable challenge. We describe the methods utilized in the Structural Genomics Consortium (SGC) in Oxford, resulting in successful purification of 48% of human proteins attempted; of those, the structures of ∼40% were solved by X-ray crystallography. The main driver has been the parallel processing of multiple (typically 9–20) truncated constructs of each target; modest diversity in vectors and host systems; and standardized purification procedures. We provide method details as well as data on the properties of the constructs leading to crystallized proteins and the impact of methodological variants. These can be used to formulate guidelines for initial approaches to expression of new eukaryotic proteins.

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.

Background: BTB-Kelch proteins, including KLHL11, are proposed to bind Cul3 through a “3-box” motif to form E3 ubiquitin ligases.Results: We solved crystal structures of the KLHL11-Cul3 complex and four Kelch domains.Conclusion: The 3-box forms a hydrophobic groove that binds a specific N-terminal extension of Cul3.Significance: Dimeric BTB-Kelch proteins bind two Cul3 molecules and support a two-site model for substrate recognition.

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD ؉ to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD ؉ -dependent deacetylation.Macrodomains are evolutionarily conserved structural domains found in proteins with diverse cellular functions (1, 2). Prior evidence suggested that macrodomains function as binding modules of NAD ϩ metabolites, including ADP-ribose/ poly(ADP-ribose) (3-7) and O-acetyl-ADP-ribose (OAADPr) 5 (8, 9). OAADPr is produced in reactions catalyzed by NAD ϩ -dependent protein/histone deacetylases (10, 11), which regulate gene silencing, metabolic enzymes, life span, and many other cellular processes (12)(13)(14). OAADPr has been implicated as a signaling molecule, modulating cellular processes affected by NAD ϩ -dependent protein/histone deacetylation (15-17). The binding of OAADPr and other NAD ϩ metabolites to macrodomains such as the histone variant macroH2A1.1 (1, 5, 8, 9) suggests a possible connection between metabolic regulation, gene activity, and chromatin structure. The mechanism by which cells regulate and utilize OAADPr is not well understood. In yeast, the NUDIX ADP-ribose pyrophosphatase Ysa1 modulates the cellular levels of both ADPr and OAADPr, converting each to AMP and the corresponding ribose-phosphate (17). Cells lacking ysa1 exhibit an increased resistance to oxidative insults and produce lower levels of endogenous reactive oxygen species. In vitro, the poly (ADP-ribose) glycohydrolase ARH3 was capable of removing the acetyl group from OAADPr (18), though the activity was orders of magnitude slower than that observed for the NUDIX family (19). In eukaryotic cell extracts at least two different cellular activities were shown to contribute to OAADPr deacetylation (19), but the identities of these factors remain unknown.Here we report a direct functional connection between sirtuins and a family of macrodomain proteins. We demonstrate that proteins belonging to this distinct branch of macrodomains are OAADPr deacetylases that...

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

RecQ-like helicases, which include 5 members in the human genome, are important in maintaining genome integrity. We present a crystal structure of a truncated form of the human RECQ1 protein with Mg-ADP. The truncated protein is active in DNA fork unwinding but lacks other activities of the full-length enzyme: disruption of Holliday junctions and DNA strand annealing. The structure of human RECQ1 resembles that of Escherichia coli RecQ, with some important differences. All structural domains are conserved, including the 2 RecA-like domains and the RecQ-specific zinc-binding and winged-helix (WH) domains. However, the WH domain is positioned at a different orientation from that of the E. coli enzyme. We identify a prominent ␤-hairpin of the WH domain as essential for DNA strand separation, which may be analogous to DNA strand-separation features of other DNA helicases. This hairpin is significantly shorter in the E. coli enzyme and is not required for its helicase activity, suggesting that there are significant differences between the modes of action of RecQ family members.DNA helicase ͉ DNA repair ͉ Holliday junction ͉ structural genomics ͉ winged helix T he RecQ helicases are a family of DNA-unwinding enzymes conserved from prokaryotes to mammals that play a key role in the maintenance of genome stability. The RecQ helicase family has 5 representatives in the human genome (1-3): RECQ1 (also known as RECQL or RECQL1), BLM, WRN, RECQ4, and RECQ5. Although these 5 enzymes are similar in their catalytic core, they probably have distinct functions, as indicated by the genetic disorders associated to mutations in the genes of BLM, WRN, and RECQ4. In particular, mutations in the gene encoding for BLM (4) are associated with the Bloom's syndrome (BS), which is manifested as an increased incidence of a wide spectrum of cancers. Werner's syndrome (WS), which is linked to mutations in the WRN (5) gene, involves many signs of premature aging, as well as a predisposition to a more limited spectrum of cancers. Mutations in the gene of RECQ4 are the cause of more varied genetic disease phenotypes, including Rothmund-Thomson (RTS) (6, 7), RAPADILINO (8), and Baller-Gerold (9) syndromes. No disease phenotypes have been associated with mutations in the genes of the other 2 family members, RECQ1 and RECQ5 yet, although they may be responsible for additional cancer predisposition disorders that are distinct from RTS, BS, and WS. In this regard, interesting candidates are patients with a phenotype similar to that of RTS individuals who do not carry any mutations in the RECQ4 gene (7). A possible role of RECQ1 in genome maintenance is suggested by several observations (reviewed in ref 10). Biochemical purification from human embryonic kidney cells recovered RECQ1 as the major Holliday junction (HJ) branch migration activity (11). Knockout of the RECQ1 gene in mice (12) or suppression of its expression in HeLa cells (11) resulted in cellular phenotypes that include chromosomal instability, increased sister chromatid exchange, and hei...