Oxidative DNA damage on the VEGF G-quadruplex forming promoter is repaired via long-patch BER.

In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays.

A two-residue nascent-strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA.

Steric exclusion is a key element of enzyme substrate specificity, including in polymerases. Such substrate specificity restricts the enzymatic synthesis of 2'-modified nucleic acids, which are of interest in nucleic-acid-based drug development. Here we describe the discovery of a two-residue, nascent-strand, steric control 'gate' in an archaeal DNA polymerase.

Processing oxidatively damaged bases at DNA strand breaks by APE1.

Reactive oxygen species attack the structure of DNA, thus altering its base-pairing properties. Consequently, oxidative stress-associated DNA lesions are a major source of the mutation load that gives rise to cancer and other diseases. Base excision repair (BER) is the pathway primarily tasked with repairing DNA base damage, with apurinic/apyrimidinic endonuclease (APE1) having both AP-endonuclease and 3' to 5' exonuclease (exo) DNA cleavage functions.

Construction of a Three-Color Prism-Based TIRF Microscope to Study the Interactions and Dynamics of Macromolecules.

Single-molecule total internal reflection fluorescence (TIRF) microscopy allows for the real-time visualization of macromolecular dynamics and complex assembly. Prism-based TIRF microscopes (prismTIRF) are relatively simple to operate and can be easily modulated to fit the needs of a wide variety of experimental applications. While building a prismTIRF microscope without expert assistance can pose a significant challenge, the components needed to build a prismTIRF microscope are relatively affordable and, with some guidance, the assembly can be completed by a determined novice.

History of DNA polymerase β X-ray crystallography.

DNA polymerase β (Pol β) is an essential mammalian enzyme involved in the repair of DNA damage during the base excision repair (BER) pathway. In hopes of faithfully restoring the coding potential to damaged DNA during BER, Pol β first uses a lyase activity to remove the 5'-deoxyribose phosphate moiety from a nicked BER intermediate, followed by a DNA synthesis activity to insert a nucleotide triphosphate into the resultant 1-nucleotide gapped DNA substrate. This DNA synthesis activity of Pol β has served as a model to characterize the molecular steps of the nucleotidyl transferase mechanism used by mammalian DNA polymerases during DNA synthesis.

AP-endonuclease 1 sculpts DNA through an anchoring tyrosine residue on the DNA intercalating loop.

Base excision repair (BER) maintains genomic stability through the repair of DNA damage. Within BER, AP-endonuclease 1 (APE1) is a multifunctional enzyme that processes DNA intermediates through its backbone cleavage activity. To accomplish these repair activities, APE1 must recognize and accommodate several diverse DNA substrates.

Molecular and structural characterization of disease-associated APE1 polymorphisms.

Under conditions of oxidative stress, reactive oxygen species (ROS) continuously assault the structure of DNA resulting in oxidation and fragmentation of the nucleobases. When the nucleobase structure is altered, its base-pairing properties may also be altered, promoting mutations. Consequently, oxidative DNA damage is a major source of the mutation load that gives rise to numerous human maladies, including cancer.

Functions of the major abasic endonuclease (APE1) in cell viability and genotoxin resistance.

DNA is susceptible to a range of chemical modifications, with one of the most frequent lesions being apurinic/apyrimidinic (AP) sites. AP sites arise due to damage-induced (e.g.

Structure and function relationships in mammalian DNA polymerases.

DNA polymerases are vital for the synthesis of new DNA strands. Since the discovery of DNA polymerase I in Escherichia coli, a diverse library of mammalian DNA polymerases involved in DNA replication, DNA repair, antibody generation, and cell checkpoint signaling has emerged. While the unique functions of these DNA polymerases are differentiated by their association with accessory factors and/or the presence of distinctive catalytic domains, atomic resolution structures of DNA polymerases in complex with their DNA substrates have revealed mechanistic subtleties that contribute to their specialization.

Propagation of the Allosteric Signal in Phosphofructokinase from Examined by Methyl-Transverse Relaxation-Optimized Spectroscopy Nuclear Magnetic Resonance.

Phosphofructokinase from (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose 6-phosphate (Fru-6-P), is allosterically regulated by the K-type inhibitor phosphoenolpyruvate (PEP). The allosteric coupling between the substrate and inhibitor is quantified by a standard coupling free energy that defines an equilibrium with the Fru-6-P-bound and PEP-bound complexes on one side and the apo form and ternary complex on the other.

APE1: A skilled nucleic acid surgeon.

Before a deleterious DNA lesion can be replaced with its undamaged counterpart, the lesion must first be removed from the genome. This process of removing and replacing DNA lesions is accomplished by the careful coordination of several protein factors during DNA repair. One such factor is the multifunctional enzyme human apurinic/apyrimidinic endonuclease 1 (APE1), known best for its DNA backbone cleavage activity at AP sites during base excision repair (BER).

Apurinic/apyrimidinic (AP) endonuclease 1 processing of AP sites with 5' mismatches.

Despite the DNA duplex being central to biological functions, many intricacies of this molecule, including the dynamic nature of mismatched base pairing, are still unknown. The unique conformations adopted by DNA mismatches can provide insight into the forces at play between nucleotides. Moreover, DNA-binding proteins apply their own individualized steric and electrochemical influences on the nucleotides that they interact with, further altering base-pairing conformations.

Molecular snapshots of APE1 proofreading mismatches and removing DNA damage.

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair enzyme which uses a single active site to process DNA damage via two distinct activities: (1) AP-endonuclease and (2) 3' to 5' exonuclease. The AP-endonuclease activity cleaves at AP-sites, while the exonuclease activity excises bulkier 3' mismatches and DNA damage to generate clean DNA ends suitable for downstream repair. Molecular details of the exonuclease reaction and how one active site can accommodate various toxic DNA repair intermediates remains elusive despite being biologically important.

Capturing a mammalian DNA polymerase extending from an oxidized nucleotide.

The oxidized nucleotide, 8-oxo-7,8-dihydro-2΄-deoxyguanosine (8-oxoG), is one of the most abundant DNA lesions. 8-oxoG plays a major role in tumorigenesis and human disease. Biological consequences of 8-oxoG are mediated in part by its insertion into the genome, making it essential to understand how DNA polymerases handle 8-oxoG.

Base excision repair of oxidative DNA damage: from mechanism to disease.

Reactive oxygen species continuously assault the structure of DNA resulting in oxidation and fragmentation of the nucleobases. Both oxidative DNA damage itself and its repair mediate the progression of many prevalent human maladies. The major pathway tasked with removal of oxidative DNA damage, and hence maintaining genomic integrity, is base excision repair (BER).

The effect of introducing small cavities on the allosteric inhibition of phosphofructokinase from Bacillus stearothermophilus.

The allosteric coupling free energy between ligands fructose-6-phosphate (Fru-6-P) and phospho(enol)pyruvate (PEP) for phosphofructokinase-1 (PFK) from the moderate thermophile, Bacillus stearothermophilus (BsPFK), results from compensating enthalpy and entropy components. In BsPFK the positive coupling free energy that defines inhibition is opposite in sign from the negative enthalpy term and is therefore determined by the larger absolute value of the negative entropy term. Variants of BsPFK were made to determine the effect of adding small cavities to the structure on the allosteric function of the enzyme.