RESUMEN

This work provides a procedure and database for obtaining the vibrational frequency scale factors that align quantum chemically computed harmonic frequencies with experimental vibrational spectroscopic data. The database comprises 441 molecules of various sizes, from diatomics to the buckminsterfullerene C60. We provide scale factors for 27 dispersion-corrected methods, 24 of which are DF-Dn/B with DF=BLYP, PBE, B3LYP, PBE0, Dn=D3(BJ), D4, and B=6-31G, def2-SVP, def2-TZVP, and three of them are the 3c-family composite methods (HF-3c, PBEh-3c, and r2$SCAN-3c). The two scale factors are derived for each method: the absolute scaling, minimizing the absolute deviation of the scaled harmonic frequency from the experimental value, and the relative scaling, which minimizes an analogous relative deviation. The absolute type of scaling is recommended for frequencies above 2000 cm-1, while the relative scaling is optimal for frequencies below 2000 cm-1.

RESUMEN

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme-DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme-DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.

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RESUMEN

The base excision repair (BER) pathway consists of sequential action of DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease necessary to remove a damaged base and generate a single-strand break in duplex DNA. Human multifunctional AP endonuclease 1 (APE1, a.k.a. APEX1, HAP-1, or Ref-1) plays essential roles in BER by acting downstream of DNA glycosylases to incise a DNA duplex at AP sites and remove 3'-blocking sugar moieties at DNA strand breaks. Human 8-oxoguanine-DNA glycosylase (OGG1), methyl-CpG-binding domain 4 (MBD4, a.k.a. MED1), and alkyl-N-purine-DNA glycosylase (ANPG, a.k.a. Aag or MPG) excise a variety of damaged bases from DNA. Here we demonstrated that the redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NΔ61) cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates. Electron microscopy imaging of APE1-DNA complexes revealed oligomerization of APE1 along the DNA duplex and APE1-mediated DNA bridging followed by DNA aggregation. APE1 polymerizes on both undamaged and damaged DNA in cooperative mode. Association of APE1 with undamaged DNA may enable scanning for damage; however, this event reduces effective concentration of the enzyme and subsequently decreases APE1-catalyzed cleavage rates on long DNA substrates. We propose that APE1 oligomers on DNA induce helix distortions thereby enhancing molecular recognition of DNA lesions by DNA glycosylases via a conformational proofreading/selection mechanism. Thus, APE1-mediated structural deformations of the DNA helix stabilize the enzyme-substrate complex and promote dissociation of human DNA glycosylases from the AP site with a subsequent increase in their turnover rate. SIGNIFICANCE STATEMENT: The major human apurinic/apyrimidinic (AP) endonuclease, APE1, stimulates DNA glycosylases by increasing their turnover rate on duplex DNA substrates. At present, the mechanism of the stimulation remains unclear. We report that the redox domain of APE1 is necessary for the active mode of stimulation of DNA glycosylases. Electron microscopy revealed that full-length APE1 oligomerizes on DNA possibly via cooperative binding to DNA. Consequently, APE1 shows DNA length dependence with preferential repair of short DNA duplexes. We propose that APE1-catalyzed oligomerization along DNA induces helix distortions, which in turn enable conformational selection and stimulation of DNA glycosylases. This new biochemical property of APE1 sheds light on the mechanism of redox function and its role in DNA repair.

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RESUMEN

In all organisms, DNA glycosylases initiate base excision repair pathways resulting in removal of aberrant bases from DNA. Human SMUG1 belongs to the superfamily of uracil-DNA glycosylases catalyzing the hydrolysis of the N-glycosidic bond of uridine and uridine lesions bearing oxidized groups at C5: 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), and 5-hydroxyuridine (5hoU). An apurinic/apyrimidinic (AP) site formed as the product of an N-glycosylase reaction is tightly bound to hSMUG1, thus inhibiting the downstream action of AP-endonuclease APE1. The steady-state kinetic parameters (kcat and KM; obtained from the literature) correspond to the enzyme turnover process limited by the release of hSMUG1 from the complex with the AP-site. In the present study, our objective was to carry out a stopped-flow fluorescence analysis of the interaction of hSMUG1 with a DNA substrate containing a dU:dG base pair to follow the pre-steady-state kinetics of conformational changes in both molecules. A comparison of kinetic data obtained by means of Trp and 2-aminopurine fluorescence and Förster resonance energy transfer (FRET) detection allowed us to elucidate the stages of specific and nonspecific DNA binding, to propose the mechanism of damaged base recognition by hSMUG1, and to determine the true rate of the catalytic step. Our results shed light on the kinetic mechanism underlying the initiation of base excision repair by hSMUG1 using the "wedge" strategy for DNA lesion search.

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