On the role of proofreading exonuclease in bypass of a 1,2 d(GpG) cisplatin adduct by the herpes simplex virus-1 DNA polymerase.
UL30, the herpes simplex virus type-1 DNA polymerase, stalls at the base preceding a cisplatin crosslinked 1,2 d(GpG) dinucleotide and engages in a futile cycle of incorporation and excision by virtue of its 3'-5' exonuclease. Therefore, we examined the translesion synthesis (TLS) potential of an exonuclease-deficient UL30 (UL30D368A). We found that UL30D368A did not perform complete translesion synthesis but incorporated one nucleotide opposite the first base of the adduct. This addition was affected by the propensity of the enzyme to dissociate from the damaged template. Consequently, addition of the polymerase processivity factor, UL42, increased nucleotide incorporation opposite the lesion. The addition of Mn(2+), which was previously shown to support translesion synthesis by wild-type UL30, also enabled limited bypass of the adduct by UL30D368A. We show that the primer terminus opposite the crosslinked d(GpG) dinucleotide and at least three bases downstream of the lesion is unpaired and not extended by the enzyme. These data indicate that the primer terminus opposite the lesion may be sequestered into the exonuclease site of the enzyme. Consequently, elimination of exonuclease activity alone, without disrupting binding, is insufficient to permit bypass of a bulky lesion by this enzyme.
Arana
Mercedes E
ME
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101, USA.
Song
Liping
L
Tanguy Le Gac
Nicolas
N
Parris
Deborah S
DS
Villani
Giuseppe
G
Boehmer
Paul E
PE
D002945:Cisplatin / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000494:pharmacology*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017895:Manganese Compounds / Q000494:pharmacology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D015226:Dinucleoside Phosphates; D017895:Manganese Compounds; D014764:Viral Proteins; C094059:cisplatin-deoxy(guanosine monophosphate guanosine) adduct; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus; D002945:Cisplatin