Effect of manganese on in vitro replication of damaged DNA catalyzed by the herpes simplex virus type-1 DNA polymerase.
In vitro bypass of damaged DNA by replicative DNA polymerases is usually blocked by helix-distorting or bulky DNA lesions. In this study, we report that substitution of the divalent metal ion Mg2+ with Mn2+ promotes quantitative replication of model DNA substrates containing the major cisplatin or N-2-acetylaminofluorene adducts by the catalytic subunit (UL30) of the replicative DNA polymerase of herpes simplex virus. The ability of Mn2+ ions to confer bypass of bulky lesions was not observed with other replicative DNA polymerases of the B family, such as bacteriophage T4 or delta polymerases. However, for these enzymes, manganese induced the incorporation of one nucleotide opposite the first (3') guanine of the d(GpG) intrastrand cisplatin lesion. Translesion replication of the cisplatin adduct by UL30 led to the incorporation of mismatched bases, with the preferential incorporation of dAMP opposite the 3' guanine of the lesion. Furthermore, substitution of MgCl2 with MnCl2 greatly inhibited the 3' to 5' exonuclease of UL30 but had a far lesser effect on that of T4 DNA polymerase. Finally, manganese induced a conformational change in the structure of UL30 bound to the platinated substrate. Taken together, the latter findings suggest a mechanism by which manganese might allow UL30 to efficiently promote translesion DNA synthesis in vitro.
Laboratoire de Pharmacologie et de Biologie Structurale, CNRS-UMR 5089, 205 route de Narbonne, 31077 Toulouse cedex 4, France. giuseppe.villani@ipbs.fr
Giuseppe
G
Villani
Nicolas
N
Tanguy Le Gac
Luc
L
Wasungu
Dominique
D
Burnouf
Robert P
RP
Fuchs
Paul E
PE
Boehmer
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology*; D002945:Cisplatin / Q000737:chemistry; D018736:DNA Adducts / Q000737:chemistry; D004249:DNA Damage; D004258:DNA Polymerase III / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology*; D011487:Protein Conformation; D014764:Viral Proteins / Q000378:metabolism
D002712:Chlorides; D018736:DNA Adducts; D015226:Dinucleoside Phosphates; D017895:Manganese Compounds; D014764:Viral Proteins; C094059:cisplatin-deoxy(guanosine monophosphate guanosine) adduct; C069983:gene 43 protein, Enterobacteria phage T4; D015636:Magnesium Chloride; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus; D002945:Cisplatin; C025340:manganese chloride