Translesional synthesis on DNA templates containing the 2′-deoxyribonolactone lesion

A site-specifically modified oligonucleotide containing a single 2′-deoxyribonolactone lesion was used as a template for primer extension reactions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo(+)) or deficient (KF exo(–)) in exonuclease activity. Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2′-deoxyribonolactone. KF exo(–) preferentially incorporated nucleotides opposite the lesion following the frequency order dAMP > dGMP >> dTMP ~ dCMP and thus appeared to obey the ‘A rule’ for preferential incorporation as has been shown previously for the 2′-deoxyribose abasic site. In the sequence context examined, the primer extension by KF exo(–) appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP. These two nucleotides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations. We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo(–) and thus the miscoding and mutational potential of the 2′-deoxyribonolactone in E.coli.