Fidelity of Escherichia coli DNA polymerase IV. Preferential generation of small deletion mutations by dNTP-stabilized misalignment.

Escherichia coli DNA polymerase IV (pol IV), a member of the error-prone Y family, predominantly generates -1 frameshifts when copying DNA in vitro. T-->G transversions and T-->C transitions are the most frequent base substitutions observed. The in vitro data agree with mutational spectra obtained when pol IV is overexpressed in vivo. Single base deletion and base substitution rates measured in the lacZalpha gene in vitro are, on average, 2 x 10(-4) and 5 x 10(-5), respectively. The range of misincorporation and mismatch extension efficiencies determined kinetically are 10(-3) to 10(-5). The presence of beta sliding clamp and gamma-complex clamp loading proteins strongly enhance pol IV processivity but have no discernible influence on fidelity. By analyzing changes in fluorescence of a 2-aminopurine template base undergoing replication in real time, we show that a "dNTP-stabilized" misalignment mechanism is responsible for making -1 frameshift mutations on undamaged DNA. In this mechanism, a dNTP substrate is paired "correctly" opposite a downstream template base, on a "looped out" template strand instead of mispairing opposite a next available template base. By using the same mechanism, pol IV "skips" past an abasic template lesion to generate a -1 frameshift. A crystal structure depicting dNTP-stabilized misalignment was reported recently for Sulfolubus solfataricus Dpo4, a Y family homolog of Escherichia coli pol IV.

Incorporation of oxidatively modified 2'-deoxynucleotide triphosphates by HIV-1 RT on RNA and DNA templates.

Oxidatively modified deoxynucleotide triphosphates (dN(oxo)TPs) present in nucleotide precursor pools may contribute to retroviral mutagenesis as a result of incorporation and ambiguous base pairing during reverse transcriptase mediated replication. We have examined the incorporation of 5-hydroxy-2'-deoxycytosine triphosphate (5-HO-dCTP) and 2'-deoxyinosine triphosphate (dITP) by HIV-1 reverse transcriptase (HIV-1 RT) on DNA and RNA templates of the same sequence in order to evaluate their mutagenic potential. Significant variations in insertion frequencies at homologous nucleotide positions were observed for each dN(oxo)TP, in general favoring the RNA template. A comparison of steady-state kinetics revealed a 10-fold preference for 5-HO-dCTP incorporation opposite G in RNA. Insertion frequencies for dITP were 2- to 20-fold greater on RNA for every base position examined. One exception to this general trend was observed for the insertion of 5-HO-dCTP by HIV-1 RT opposite A, which favored the DNA template by 4-fold. Deoxyinosine triphosphate was inserted opposite C with an 8-fold higher frequency compared to dGTP in RNA, while on DNA templates, the incorporation frequencies were equivalent. However, incorporation of dITP opposite other bases was characterized by relatively low frequencies. The RNA template bias observed for dN(oxo)TP incorporation is discussed in terms of recent efforts to utilize 5-OH-dCTP as an anti-HIV agent.