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.