Thermodynamic and in Vitro Replication Studies of an Intrastrand G[8-5]C Cross-Link Lesion

Abstract

We recently identified, from the gamma-irradiation mixture of duplex DNA, a new intrastrand G[8-5]C cross-link lesion, in which the C8 atom of guanine and the C5 atom of its 3' neighboring cytosine are covalently bonded, and carried out in vitro replication studies for the lesion-bearing substrate with a translesion synthesis polymerase, yeast polymerase eta. Here we extended the in vitro replication studies to two replicative polymerases, exonuclease-deficient bacteriophage T7 DNA polymerase (T7(-)) and HIV reverse transcriptase (HIV-RT). Primer extension assays showed that both polymerases stopped synthesis after incorporating a nucleotide opposite the 3'-cytosine in the G[8-5]C lesion. Steady-state kinetic measurements for nucleotide incorporation opposite the 3'-cytosine of the lesion showed that both T7(-) and HIV-RT preferentially incorporated the correct nucleotide, dGMP. We also examined the thermal stabilities and base pairing properties of G[8-5]C in d(ATGGCG[8-5]CGCTAT). The G[8-5]C lesion destabilizes the duplex form by approximately 4 kcal/mol in free energy at 25 degrees C relative to the undamaged parent duplex, and the thermally most stable duplex has natural bases opposite the lesion.