Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo(-) Klenow fragment of Pol I

The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Kle now fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene dol epoxide isomer [(+)-anti-BPDE] to N-2-guanine (G*). Four different 43-base long oligonucl eotide templates were constructed with G* at a site 19 bases from the 5'-en d. All bases were identical, except for the pyrimidines, X or Y, flanking G * (sequence context 5'-...XG*,, with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G*, wi th A > G). Depending on X or Y, full lesion bypass differed by factors of s imilar to1.5-5 (similar to0.6-3.0% bypass efficiencies). A downstream T fla nking G* on the 5'-side instead of C favors full lesion bypass, while an up stream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly do minant (similar to5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products (similar to0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and ( 2) extension of the primer beyond G* by a single dNTP, or in the presence o f all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* w ere investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching similar to 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are tra ced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) w ith the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influ ence DNA translesion synthesis.