Structural accommodation of ribonucleotide incorporation by the DNA repair enzyme polymerase Mu

While most DNA polymerases discriminate against ribonucleotide triphosphate (rNTP) incorporation very effectively, the Family X member DNA polymerase μ (Pol μ) incorporates rNTPs almost as efficiently as deoxyribonucleotides. To gain insight into how this occurs, here we have used X-ray crystallography to describe the structures of pre- and post-catalytic complexes of Pol μ with a ribonucleotide bound at the active site. These structures reveal that Pol μ binds and incorporates a rNTP with normal active site geometry and no distortion of the DNA substrate or nucleotide. Moreover, a comparison of rNTP incorporation kinetics by wildtype and mutant Pol μ indicates that rNTP accommodation involves synergistic interactions with multiple active site residues not found in polymerases with greater discrimination. Together, the results are consistent with the hypothesis that rNTP incorporation by Pol μ is advantageous in gap-filling synthesis during DNA double strand break repair by nonhomologous end joining, particularly in nonreplicating cells containing very low deoxyribonucleotide concentrations

Engineering sulfotransferases to modify heparan sulfate

The biosynthesis of heparan sulfate (HS), an essential glycan in many organisms, involves an array of specialized sulfotransferases. Here, we present a study aimed at engineering the substrate specificity of different HS 3-O-sulfotransferase isoforms. Based on the crystal structures, we identified a pair of amino acid residues responsible for selecting the substrates. Mutations at these residues altered the substrate specificities. Our results demonstrated the feasibility of tailoring the specificity of sulfotransferases to modify HS with desired functions

Sustained active site rigidity during synthesis by human DNA polymerase μ

DNA polymerase mu (Pol μ) is the only template-dependent human DNA polymerase capable of repairing double strand DNA breaks (DSBs) with unpaired 3′-ends in non-homologous end joining (NHEJ). To probe this function, we structurally characterized Pol μ’s catalytic cycle for single nucleotide incorporation. These structures indicate that, unlike other template-dependent DNA polymerases, there are no large-scale conformational changes in protein subdomains, amino acid side chains, or DNA upon dNTP binding or catalysis. Instead, the only major conformational change is seen earlier in the catalytic cycle, when the flexible Loop1 region repositions upon DNA binding. Pol μ variants with changes in Loop1 have altered catalytic properties and are partially defective in NHEJ. The results indicate that specific Loop1 residues contribute to Pol μ’s unique ability to catalyze template-dependent NHEJ of DSBs with unpaired 3′-ends

A comparison of BRCT domains involved in nonhomologous end-joining: Introducing the solution structure of the BRCT domain of polymerase lambda

Three of the four family X polymerases, DNA polymerase λ, DNA polymerase µ, and TdT have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase λ, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase λ BRCT domain reveals structural similarity to the BRCT domains of polymerase µ and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol λ plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol µ, Arg43. In contrast, the hydrophobic Leu60 residue of Pol λ contributes less significantly to binding than the structurally homologous Phe46 residue of Pol µ. A third leucine residue involved in the binding and activity of Pol µ, is nonconservatively replaced by a glutamine in Pol λ (Gln64) and, based on binding and activity data, is apparently unimportant for Pol λ interactions with the NHEJ complex. In conclusion, both the structure of the Pol λ BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol µ and TdT

Inhibitors of Streptococcus pneumoniae Surface Endonuclease EndA Discovered by High-Throughput Screening Using a PicoGreen Fluorescence Assay

The human commensal pathogen, Streptococcus pneumoniae, expresses a number of virulence factors that promote serious pneumococcal diseases, resulting in significant morbidity and mortality worldwide. These virulence factors may give S. pneumoniae the capacity to escape immune defenses, resist antimicrobial agents, or a combination of both. Virulence factors also present possible points of therapeutic intervention. The activities of the surface endonuclease, EndA, allow S. pneumoniae to establish invasive pneumococcal infection. EndA’s role in DNA uptake during transformation contributes to gene transfer and genetic diversitifcation. Moreover, EndA’s nuclease activity degrades the DNA backbone of neutrophil extracellular traps (NETs), allowing pneumococcus to escape host immune responses. Given its potential impact on pneumococcal pathogenicity, EndA is an attractive target for novel antimicrobial therapy. Herein, we describe the development of a high-throughput screening assay for the discovery of nuclease inhibitors. Nuclease-mediated digestion of double-stranded DNA was assessed using fluorescence intensity changes of the DNA dye ligand, PicoGreen. Under optimized conditions, the assay provided robust and reproducible activity data (Z'=0.87) and was used to screen 4727 small molecules against an imidazole-rescued variant of EndA. In total, 10 small molecules were confirmed as novel EndA inhibitors that may have utility as research tools for understanding pneumococcal pathogenesis, and ultimately drug discovery

Structural insight into the substrate specificity of DNA polymerase μ.

DNA polymerase l (Pol l) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol l's unusual substrate specificity, we describe the 2.4 Å crystal structure of the polymerase domain of murine Pol l bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol l that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol l from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3¢ ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases. To accomplish the many DNA transactions involved in stably maintaining and replicating large genomes, mammals encode 16 DNA polymerases classified in families A, B, X and Y 1 . Clues to their functions include their substrate specificity, which can differ substantially, even for members of the same family. For example, two family X enzymes with intrinsic 5¢-deoxyribose 5¢-phosphate (dRP) lyase activities, DNA polymerase b (Pol b) and Pol l, are template dependent and efficiently fill short gaps, consistent with synthesis during short patch base excision repair 2,3 . Unlike Pol b, Pol l has a BRCT domain and probably participates in NHEJ of double-strand breaks (DSBs) in DNA 4-6 . Specifically, Pol l has a role in V(D)J recombination at immunoglobulin heavy-chain loci Pol m has an unusual primer-template specificity. Like Pol b and Pol l, Pol m can fill short gaps in a template-dependent manner 12 , yet it also shares with TdT the ability to catalyze template-independent synthesis 13 . Pol m has an unusually high capacity to extend misaligned primer termini 14 ; it can perform translesion synthesis (TLS) in vitro To test the relationship between substrate specificity and physiological function, one can compare structures of these polymerases bound to primer-templates. Structures already exist for Pol b 19-22 , Pol l 23-26 and TdT 27 , but not yet for Pol m. Here we fill this knowledge gap by describing a 2.4-Å crystal structure of a ternary complex of the polymerase domain of murine Pol m. This structure reveals substrate interactions unique to Pol m, compared with Pol b, Pol l and TdT. To test whether such differences are important for unusual substrate use by Pol m, we examined the properties of the wild-type enzyme and an H329A mutant that perturbs interactions with the DNA that are not found in Pol b and Pol l. A similar substitution was performed on the homologous histidine (H342A) in human TdT. These mutations were made to test whether the histidine is important for templateindependent synthesis and for template-dependent synthesis with substrates lacking a template nucleotide at the 3¢ primer terminus. RESULTS Overall structure of a ternary Pol l-DNA-ddTTP complex The polymerization domain (Pro132-Ala496) of murine Pol m was crystallized in a ternary complex with a gapped template-primer and a correctly paired nucleoside triphosphate bound in the nascent base pair-binding pocket. The DNA contained an 11-nucleotide (nt

Structural Analysis of the Sulfotransferase (3- O -Sulfotransferase Isoform 3) Involved in the Biosynthesis of an Entry Receptor for Herpes Simplex Virus 1

Heparan sulfate (HS) plays essential roles in assisting herpes simplex virus infection and other biological processes. The biosynthesis of HS includes numerous specialized sulfotransferases that generate a variety of sulfated saccharide sequences, conferring the selectivity of biological functions of HS. We report a structural study of human HS 3-O-sulfotransferase isoform 3 (3-OST-3), a key sulfotransferase that transfers a sulfuryl group to a specific glucosamine in HS generating an entry receptor for herpes simplex virus 1. We have obtained the crystal structure of 3-OST-3 at 1.95 Å in a ternary complex with 3′-phosphoadenosine 5′-phosphate and a tetrasaccharide substrate. Mutational analyses were also performed on the residues involved in the binding of the substrate. Residues Gln255 and Lys368 are essential for the sulfotransferase activity and lie within hydrogen bonding distances to the carboxyl and sulfo groups of the uronic acid unit. These residues participate in the substrate recognition of 3-OST-3. This structure provides atomic level evidence for delineating the substrate recognition and catalytic mechanism for 3-OST-3

Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae

EndA is a sequence non-specific endonuclease that serves as a virulence factor during Streptococcus pneumoniae infection. Expression of EndA provides a strategy for evasion of the host's neutrophil extracellular traps, digesting the DNA scaffold structure and allowing further invasion by S. pneumoniae. To define mechanisms of catalysis and substrate binding, we solved the structure of EndA at 1.75 Å resolution. The EndA structure reveals a DRGH (Asp-Arg-Gly-His) motif-containing ββα-metal finger catalytic core augmented by an interesting ‘finger-loop’ interruption of the active site α-helix. Subsequently, we delineated DNA binding versus catalytic functionality using structure-based alanine substitution mutagenesis. Three mutants, H154A, Q186A and Q192A, exhibited decreased nuclease activity that appears to be independent of substrate binding. Glu205 was found to be crucial for catalysis, while residues Arg127/Lys128 and Arg209/Lys210 contribute to substrate binding. The results presented here provide the molecular foundation for development of specific antibiotic inhibitors for EndA

Extracellular volume quantification in isolated hypertension - changes at the detectable limits?

The funding source (British Heart Foundation and UK National Institute for Health Research) provided salaries for research training (FZ, TT, DS, SW), but had no role in study design, collection, analysis, interpretation, writing, or decisions with regard to publication. This work was undertaken at University College London Hospital, which received a proportion of funding from the UK Department of Health National Institute for Health Research Biomedical Research Centres funding scheme. We are grateful to King’s College London Laboratories for processing the collagen biomarker panel