Frameshift Deletion by Sulfolobus solfataricus P2 DNA Polymerase Dpo4 T239W Is Selective for Purines and Involves Normal Conformational Change Followed by Slow Phosphodiester Bond Formation

Abstract

The human DNA polymerase homolog Sulfolobus solfataricus DNA polymerase IV (Dpo4) produces "؊1" frameshift deletions while copying unmodified DNA and, more frequently, when bypassing DNA adducts. As judged by steady-state kinetics and mass spectrometry, bypass of purine template bases to produce these deletions occurred rarely but with 10-fold higher frequency than with pyrimidines. The DNA adduct 1,N 2 -etheno-2-deoxyguanosine, with a larger stacking surface than canonical purines, showed the highest frequency of formation of ؊1 frameshift deletions. Dpo4 T239W, a mutant we had previously shown to produce fluorescence changes attributed to conformational change following dNTP binding opposite cognate bases (Beckman, J. W., Wang, Q., and Guengerich, F. P. (2008) J. Biol. Chem. 283, 36711-36723), reported similar conformational changes when the incoming dNTP complemented the base following a templating purine base or bulky adduct (i.e. the "؉1" base). However, in all mispairing cases, phosphodiester bond formation was inefficient. The frequency of ؊1 frameshift events and the associated conformational changes were not dependent on the context of the remainder of the sequence. Collectively, our results support a mechanism for ؊1 frameshift deletions by Dpo4 that involves formation of active complexes via a favorable conformational change that skips the templating base, without causing slippage or flipping out of the base, to incorporate a complementary residue opposite the ؉1 base, in a mechanism previously termed "dNTP-stabilized incorporation." The driving force is attributed to be the stacking potential between the templating base and the incoming dNTP base.The integrity of the genetic code is maintained by DNA polymerases, but mutations do occur (1). Mutations can be advantageous in enabling adaptation (e.g. in microbial populations or the immune system), but the loss of genetic integrity can lead to many diseases, including cancer, atherosclerosis, and neurodegenerative diseases (1-3), as well as teratology and aging (4).In the past decade, the number of recognized DNA polymerases has expanded considerably. Of these, several have been shown to be so-called translesion DNA polymerases, DNA polymerases specializing in bypass of templating adducts that normally stall the more efficient and processive replicative DNA polymerases (1, 5, 6). However, translesion DNA polymerases have relatively low fidelity when copying undamaged DNA and are prone to cause frameshift mutations, particularly deletions that reduce the nucleotide count by 1 in newly formed DNA strands, i.e. "Ϫ1" frameshifts. Frameshifts occur in the presence of many DNA adducts and are the basis of one major type of damage in the classic "Ames test" for bacterial genotoxicity, i.e. Salmonella typhimurium TA1538 and TA98 (7). Frameshift deletions also occur with unmodified DNA, particularly while copying long runs of purines or pyrimidines (8). For example, Solfataricus acidocaldarius DNA polymerase Dbh was found to make Ϫ1 deletion frameshift mutations...

A Polymerase – Tautomeric Model for Targeted Frameshift Mutations: Deletions Formation during Error-prone or SOS Replication of Double-stranded DNA Containing cis-syn Cyclobutane Thymine Dimers

“…The second model [45,46] states that certain ( 1)-frameshifts are initiated by a nucleotide misincorporation. The model [46] has been confirmed [38,48,49]. Functional studies and information on the structures of DNA polymerases allow refinement of Streisinger's original hypothesis and offer possible explanations for why misalignment error rates during DNA synthesis can vary by 10,000-fold, depending on the DNA polymerase and the nucleotide composition, symmetry, and location of the synthesis error [47].…”

“…Overexpression of enzymes of the base excision repair pathway is known to increase the frequency of frameshift mutations [37]. Human polymerase kappa uses a classical Streisinger template-slippage mechanism to generate −1 deletions in repetitive sequences, as do the bacterial and archaeal homologues [38,39]. Frameshift-inducing mutagens can selectively induce mutations in mismatch repair-deficient cells versus mismatch repair-proficient cells.…”

Human Base Excision Repair Creates a Bias Toward −1 Frameshift Mutations

“…The Y-family polymerases are particularly prone to template slipping. For example, frequent Ϫ1 slips are observed during replication of ⑀A-DNA by human polymerases , , and (43,44) and during replication of ⑀G-DNA by Dpo4 (45,46). However, other types of damaged nucleotides are not known to induce polymerase slipping.…”

Citotoxicidade do corante dinitrofenilazo CI DB291: biotransformação, estresse oxidativo e alteração epigenética em células HepG2

“…Estudos demonstraram que estas lesões são altamente mutagênicas e que podem conduzir à carcinogênese mediada pelo processo de peroxidação lipídica (ZHANG et al, 2009;GARCIA et al, 2010).…”

“…Escherichia coli e em linhagens de células humanas, sendo observadas: AKASAKA et al, 1999;LOUREIRO et al, 2002;ZHANG et al, 2009). A magnitude dos níveis basais de etenoadutos detectados em DNA de células humanas normais é de ~20 lesões a cada 10 9 bases não modificadas, sendo observada uma boa correlação entre os dados das diferentes metodologias empregadas para a quantificação (GUICHARD et al, 1996;LEVINE et al, 2000;DOERGE et al, 2000;ROBERTS et al, 2001;NAIR et al, 2007;GARCIA et al, 2010).…”