Identification of 2-Sulfonyl/Sulfonamide Pyrimidines as Covalent Inhibitors of WRN Using a Multiplexed High-Throughput Screening Assay.

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity.

Pathways of thymidine hypermodification.

The DNAs of bacterial viruses are known to contain diverse, chemically complex modifications to thymidine that protect them from the endonuclease-based defenses of their cellular hosts, but whose biosynthetic origins are enigmatic. Up to half of thymidines in the Pseudomonas phage M6, the Salmonella phage ViI, and others, contain exotic chemical moieties synthesized through the post-replicative modification of 5-hydroxymethyluridine (5-hmdU). We have determined that these thymidine hypermodifications are derived from free amino acids enzymatically installed on 5-hmdU.

Phage-encoded ten-eleven translocation dioxygenase (TET) is active in C5-cytosine hypermodification in DNA.

TET/JBP (ten-eleven translocation/base J binding protein) enzymes are iron(II)- and 2-oxo-glutarate-dependent dioxygenases that are found in all kingdoms of life and oxidize 5-methylpyrimidines on the polynucleotide level. Despite their prevalence, few examples have been biochemically characterized. Among those studied are the metazoan TET enzymes that oxidize 5-methylcytosine in DNA to hydroxy, formyl, and carboxy forms and the euglenozoa JBP dioxygenases that oxidize thymine in the first step of base J biosynthesis.

Structure and function of the two-component flavin-dependent methanesulfinate monooxygenase within bacterial sulfur assimilation.

Methyl sulfur compounds are a rich source of environmental sulfur for microorganisms, but their use requires redox systems. The bacterial sfn and msu operons contain two-component flavin-dependent monooxygenases for dimethylsulfone (DMSO) assimilation: SfnG converts DMSO to methanesulfinate (MSI), and MsuD converts methanesulfonate (MS) to sulfite. However, the enzymatic oxidation of MSI to MS has not been demonstrated, and the function of the last enzyme of the msu operon (MsuC) is unresolved.

Insights into the Biochemistry, Evolution, and Biotechnological Applications of the Ten-Eleven Translocation (TET) Enzymes.

A tight link exists between patterns of DNA methylation at carbon 5 of cytosine and differential gene expression in mammalian tissues. Indeed, aberrant DNA methylation results in various human diseases, including neurologic and immune disorders, and contributes to the initiation and progression of various cancers. Proper DNA methylation depends on the fidelity and control of the underlying mechanisms that write, maintain, and erase these epigenetic marks.

An endogenous dAMP ligand in class Ib RNR promotes assembly of a noncanonical dimer for regulation by dATP.

The high fidelity of DNA replication and repair is attributable, in part, to the allosteric regulation of ribonucleotide reductases (RNRs) that maintains proper deoxynucleotide pool sizes and ratios in vivo. In class Ia RNRs, ATP (stimulatory) and dATP (inhibitory) regulate activity by binding to the ATP-cone domain at the N terminus of the large α subunit and altering the enzyme's quaternary structure. Class Ib RNRs, in contrast, have a partial cone domain and have generally been found to be insensitive to dATP inhibition.

Glutamate 52-β at the α/β subunit interface of class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface.

Choosing the right metal: case studies of class I ribonucleotide reductases.

Over one-third of all proteins require metallation for function (Waldron, K. J., Rutherford, J.

Bacillus subtilis class Ib ribonucleotide reductase: high activity and dynamic subunit interactions.

The class Ib ribonucleotide reductase (RNR) isolated from Bacillus subtilis was recently purified as a 1:1 ratio of NrdE (α) and NrdF (β) subunits and determined to have a dimanganic-tyrosyl radical (Mn(III)2-Y·) cofactor. The activity of this RNR and the one reconstituted from recombinantly expressed NrdE and reconstituted Mn(III)2-Y· NrdF using dithiothreitol as the reductant, however, was low (160 nmol min(-1) mg(-1)). The apparent tight affinity between the two subunits, distinct from all class Ia RNRs, suggested that B.