Structural and Kinetic Studies of the Effect of Guanine N7 Alkylation and Metal Cofactors on DNA Replication

Abstract

A wide variety of endogenous and exogenous alkylating agents attack DNA to preferentially generate N7-alkylguanine (N7-alkylG) adducts. Studies on the effect of N7-alkylG lesions on biological processes have been difficult due in part to complications arising from the chemical lability of the positively charged N7-alkylG, which can readily produce secondary lesions. To assess the effect of bulky N7-alkylG on DNA replication, we prepared chemically stable N7-benzylguanine(N7bnG)-containing DNA and evaluated nucleotide incorporation opposite the lesion by human DNA polymerase β (polβ), a model enzyme for high-fidelity DNA polymerases. Kinetic studies showed that the N7-benzyl-G lesion greatly inhibited dCTP incorporation by polβ. The crystal structure of polβ incorporating dCTP opposite N7bnG showed a Watson-Crick N7bnG:dCTP. The polβ-N7bnG:dCTP structure showed an open protein conformation, a relatively disordered dCTP, and lack of catalytic metal, which explained the inefficient nucleotide incorporation opposite N7bnG. This indicates that polβ is sensitive to major groove adducts in the templating-base side and deters nucleotide incorporation opposite bulky N7-alkylG adducts by adopting a catalytically incompetent conformation. Substituting Mg2+ for Mn2+ induced an open-to-closed conformational change due to the presence of catalytic metal and stably bound dCTP and increased the catalytic efficiency by ~10-fold, highlighting the effect of binding of incoming nucleotide and catalytic metal on protein conformation and nucleotidyl transfer reaction. Overall, these results suggest that, although bulky alkyl groups at guanine-N7 may not alter base-pairing properties of guanine, the major-groove-positioned lesions in the template could impede nucleotidyl transfer by some DNA polymerases.

Identification of HMG-box family establishes the significance of SOX6 in the malignant progression of glioblastoma

“…All the mechanism studies are aimed at finding drug targets, better prevention and treatment of diseases, there have been many studies such as a large number of alkylating anticancer agents and mutagens, and might be related to DNA replication [35][36][37]. As the HMG-box proteins influence DNA-dependent processes (transcription, replication, and DNA repair) [3], DE HMG-box genes and related DE-lncRNA / DE-miRNA in GBM, might serve as a potential drug target for DNA loss repair.…”

CpRu Complexes Containing Water Soluble Phosphane PTA and Natural Purines Adenine, Guanine and Theophylline: Synthesis, Characterization, and Antiproliferative Properties

“…The complex 5 was synthesized by a procedure similar to that for 4 and therefore a similar complex was expected but containing guaninate instead of adeninate. The IR spectrum of 5 showed the typical bands for coordinated guaninate (δ(NH 2 )= 1672 (s); ν(C=O)=1608 (s); ν(C=C+C=N)=1564 (m)), [72][73][74] and its 1 H NMR spectrum displays the characteristic signals for PPh 3 , PTA and Cp in a piano-stool configuration. [65] The guaninate signals arise at 5.86 ppm (NH 2 ) and 7.14 ppm (C8-H), which are similar to those found for 2 (δ(C8-H) = 7.35 ppm).…”

Genetic variant of COL11A2 gene is functionally associated with developmental dysplasia of the hip in Chinese Han population

“…GDF5, TBX4, ASPN, PAPPA2 and TGFB1) were discovered by association study in Chinese and Caucasian populations [9][10][11][12][13]. Gene mutations not only confer the sequence changes, but can also elicit certain structural change in the microenvironment [14,15]. A variety of environmental factors have also been predicted to participate in DDH [16].…”