A 44 nucleotide DNA template containing a single site-specifically placed cisplatin adduct (cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]]) was annealed with a primer, positioning its 3'-end four bases before the adduct in the template strand. DNA polymerization in the presence of all four nucleotides revealed that both HIV-1 reverse transcriptase (RT) and T7 DNA polymerase strongly paused at one nucleotide preceding the first platinated guanine and at the positions opposite the two platinated guanines. Analysis of single nucleotide incorporation at each pause site showed that polymerization occurs with biphasic kinetics. A small percentage of DNA was bound productively, providing a small amplitude (1-3%) of a fast phase of polymerization, whereas most of the bound DNA (1-34%) was positioned at the pause site in a nonproductive manner and therefore elongated slowly (0.04-0.06 s-1). DNA substrates annealed to the cisplatin-modified template bind to HIV-1 RT with an affinity (10-20 nM) similar to that of unmodified substrates (6-9 nM). The cisplatin-DNA cross-link moderately weakened DNA binding to T7 DNA polymerase (12-115 nM) but significantly slowed the rate of incorporation of the next nucleotide (2-7 s-1 ), with larger effects closer to the cisplatin-DNA adduct. The crystal structure of the same cisplatin-DNA adduct [Takahara, P. M., Frederick, C. A., and Lippard, S. J. (1996) J. Am. Chem. Soc. 118, 12309-12321] reveals not only the bent DNA duplex but also the propeller twisted base pairs near the cisplatin-DNA adduct. The twisted base pairs may cause misalignment of the cisplatin-modified DNA at the binding cleft of T7 DNA polymerase and significantly slow the rate of the protein conformational change preceding polymerization, leading to the slight accumulation of intermediates within five base pairs of the adduct. The ground-state binding of the next correct nucleotide to the enzyme.DNA complex was weakened by the adduct with T7 DNA polymerase but unchanged with HIV-1 RT at sites other than the three strong pause sites. Nucleotide binding to both enzymes at the three strong pause sites was significantly weaker and less selective.