Mutagenesis of 8-Oxoguanine Adjacent to an Abasic Site in Simian Kidney Cells: Tandem Mutations and Enhancement of G→T Transversions

Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided >60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a >30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the AP site. While the preference for targeted base substitutions at the GUG site was T ≫ C > A > G, the same at the GUC site was T ≫ A > C > G. We conclude that base incorporation opposite an AP site is sequence-dependent. For 8-oxoG, as compared to 23−24% G→T mutants from a single 8-oxoG in a TG8-oxoT sequence context, the tandem lesions UG8-oxoT and TG8-oxoU generated ∼60 and >85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G8-oxo→T transversions that included both single and tandem mutations at the TG8-oxoU site was nearly 60% relative to about 30% at the UG8-oxoT site. A higher mutational frequency at the TG8-oxoU sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the Tm for the 8-oxoG:C pair in the TG8-oxo(AP*) sequence in a 12-mer was lower than the same in a (AP*)G8-oxoT 12-mer with ΔΔG 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G8-oxoT 12-mer duplex with a ΔΔG of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG8-oxo(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG8-oxo(AP*) sequence relative to (AP*)G8-oxoT. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG8-oxo(AP*) sequence context

Genetic Requirement for Mutagenesis of the G[8,5-Me]T Cross-Link in <i>Escherichia coli</i>: DNA Polymerases IV and V Compete for Error-Prone Bypass

γ-Radiation generates a variety of complex lesions in DNA, including the G[8,5-Me]T intrastrand cross-link in which C8 of guanine is covalently linked to the 5-methyl group of the 3′-thymine. We have investigated the toxicity and mutagenesis of this lesion by replicating a G[8,5-Me]T-modified plasmid in <i>Escherichia coli</i> with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these DNA polymerases. In the single-polymerase knockout strains, viability was the lowest in a pol V-deficient strain, which suggests that pol V is most efficient in bypassing this lesion. Most mutations were single-base substitutions or deletions, though a small population of mutants carrying two point mutations at or near the G[8,5-Me]T cross-link was also detected. Mutations in the progeny occurred at the cross-linked bases as well as at bases near the lesion site, but the mutational spectrum varied on the basis of the identity of the DNA polymerase that was knocked out. Mutation frequency was the lowest in a strain that lacked the three SOS DNA polymerases. We determined that pol V is required for most targeted G → T transversions, whereas pol IV is required for the targeted T deletions. Our results suggest that pol V and pol IV compete to carry out error-prone bypass of the G[8,5-Me]T cross-link

Synthesis and Characterization of Oligodeoxynucleotides Containing the Major DNA Adducts Formed by 1,6- and 1,8-Dinitropyrene

An efficient method for the synthesis of oligonucleotides containing a site-specific DNA adduct formed by the carcinogens 1,6- and 1,8-dinitropyrene has been developed. Palladium-catalyzed amination provided a straightforward route for the synthesis of aminonitropyrenes which, following separation, were reduced to the nitrosonitropyrenes. The N-hydroxyaminonitropyrene, generated in situ from each nitrosonitropyrene, was used successfully to introduce the dinitropyrene-derived DNA adduct at a defined site in an oligonucleotide

Percent mutations induced by Z in GZGTC and GTGZC sequence contexts normalized by TLS (i.e., % MF multiplied with TLS frequency in hundredths) for GZGTC and GTGZC constructs in HEK 293T cells without or with siRNA knockdowns of pol ζ and Rev1.

<p>Percent mutations induced by Z in <u>GZG</u>TC and GT<u>GZC</u> sequence contexts normalized by TLS (i.e., % MF multiplied with TLS frequency in hundredths) for <u>GZG</u>TC and GT<u>GZC</u> constructs in HEK 293T cells without or with siRNA knockdowns of pol ζ and Rev1.</p

Representative gel images of siRNA knockdown of TLS pols in HEK293T cells.

<p>RT-PCR shows the efficiency of inhibition of the TLS pols. M, the DNA size marker; NC, negative control siRNA. Panel (a) shows the RT-PCR of Rev1 as R1; Panel (b) shows the same for Rev3, the catalytic subunit of pol ζ (R3). In each case, as a negative control of RT-PCR, the effects of siRNA were also inspected on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. The mRNA expressions of Rev1 and Rev3 of pol ζ relative to negative control (NC) are shown in the bar graph on the bottom.</p

TLS frequencies for GZGTC and GTGZC constructs in wild type and pol II-, pol IV-, pol V-, and triple-knockout <i>E. coli</i> strains without (−) and with (+) SOS.

<p>TLS frequencies for <u>GZG</u>TC and GT<u>GZC</u> constructs in wild type and pol II-, pol IV-, pol V-, and triple-knockout <i>E. coli</i> strains without (−) and with (+) SOS.</p

A general scheme for construction of the lesion-containing vector, its replication in <i>E. coli</i> or HEK 293T cells, and analysis of the progeny.

<p>A general scheme for construction of the lesion-containing vector, its replication in <i>E. coli</i> or HEK 293T cells, and analysis of the progeny.</p

A comparison of the frequency of Z→T versus targeted Z deletion (i.e., Z→Δ) normalized by TLS (i.e., % MF multiplied with TLS frequency in hundredths) for GZGTC and GTGZC constructs in wild type and pol II-, pol IV-, pol V-, and TKO <i>E. coli</i> strains without (−) and with (+) SOS.

<p>A comparison of the frequency of Z→T versus targeted Z deletion (i.e., Z→Δ) normalized by TLS (i.e., % MF multiplied with TLS frequency in hundredths) for <u>GZG</u>TC and GT<u>GZC</u> constructs in wild type and pol II-, pol IV-, pol V-, and TKO <i>E. coli</i> strains without (−) and with (+) SOS.</p

Effects of siRNA knockdowns of pol ζ and Rev1 on the extent of replicative bypass of Z for GZGTC and GTGZC constructs.

<p>Percent TLS in the pol knockdowns was measured using an internal control of unmodified plasmid containing a different sequence near the lesion site. When control siRNA was used, the % bypass remained the same as in HEK 293T cells.</p