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

Biochemistry. 2001 Jun 5;40(22):6660-9. doi: 10.1021/bi010005o.


The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., 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, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% 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 of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with 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* influence DNA translesion synthesis.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / chemistry
  • 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / metabolism*
  • Base Sequence
  • Carcinogens, Environmental / chemistry
  • Carcinogens, Environmental / metabolism
  • Catalysis
  • DNA Damage* / genetics
  • DNA Polymerase I / chemistry
  • DNA Polymerase I / metabolism*
  • DNA Primers / isolation & purification
  • DNA Primers / metabolism
  • DNA Replication* / genetics
  • Deoxyadenine Nucleotides / metabolism
  • Deoxycytosine Nucleotides / metabolism
  • Deoxyguanine Nucleotides / metabolism
  • Deoxyguanosine / metabolism
  • Guanine / metabolism
  • Kinetics
  • Mutagenesis
  • Mutagens / chemistry
  • Mutagens / metabolism
  • Polydeoxyribonucleotides / chemistry
  • Polydeoxyribonucleotides / metabolism*
  • Pyrimidine Nucleotides / metabolism
  • Sequence Analysis, DNA / methods
  • Templates, Genetic
  • Thymine Nucleotides / metabolism


  • Carcinogens, Environmental
  • DNA Primers
  • Deoxyadenine Nucleotides
  • Deoxycytosine Nucleotides
  • Deoxyguanine Nucleotides
  • Mutagens
  • Polydeoxyribonucleotides
  • Pyrimidine Nucleotides
  • Thymine Nucleotides
  • benz(a)pyrene-7,8-dihydrodiol-9,10-epoxide poly((dT-dC).(dG-dA)) adduct
  • 2'-deoxycytidine 5'-triphosphate
  • 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
  • Guanine
  • deoxyguanosine triphosphate
  • DNA Polymerase I
  • Deoxyguanosine
  • 2'-deoxyadenosine triphosphate
  • thymidine 5'-triphosphate