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 approximately 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.
Berthet
N
N
LEDSS, Chimie Bioorganique, UMR CNRS 5616, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France. nathalie.berthet@ujf-grenoble.fr
Roupioz
Y
Y
Constant
J F
JF
Kotera
M
M
Lhomme
J
J
D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D004256:DNA Polymerase I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D008979:Moloney murine leukemia virus / Q000201:enzymology; D016296:Mutagenesis / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013400:Sugar Acids / Q000378:metabolism*; D013698:Templates, Genetic
D009711:Nucleotides; D009841:Oligonucleotides; D013400:Sugar Acids; C056405:2,4,5-trihydroxypentanoic acid gamma-lactone; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases