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 much greater than dTMP similar to 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.