Single d(GpG)/cis-diammineplatinum(II) adduct-induced inhibition of DNA polymerization

A 44 nucleotide DNA template containing a single site-specifically placed c isplatin adduct (cis-[Pt(NH3)(2){d(cpG)-N7(1),-N7(2)}]) was annealed with a primer, positioning its 3'-end four bases before the adduct in the templat e strand. DNA polymerization in the presence of all four nucleotides reveal ed that both HIV-1 reverse transcriptase (RT) and T7 DNA polymerase strongl y paused at one nucleotide preceding the first platinated guanine and at th e positions opposite the two platinated guanines, Analysis of single nucleo tide incorporation at each pause site showed that polymerization occurs wit h biphasic kinetics. A small percentage of DNA was bound productively, prov iding a small amplitude (1-3%) Of a fast phase of polymerization, whereas m ost of the bound DNA (1-34%) was positioned at the pause site in a nonprodu ctive manner and therefore elongated slowly (0.04-0.06 s(-1)). DNA substrat es annealed to the cisplatin-modified template bind to HIV-1 RT with an aff inity (10-20 nM) similar to that of unmodified substrates (6-9 nM). The cis platin-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 add uct, The crystal structure of the same cisplatin-DNA adduct [Takahara, P. M ., Frederick, C. A., and Lippard, S. J. (1996) J. Am. Chem. Sec. 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 ch ange preceding polymerization, leading to the slight accumulation of interm ediates within five base pairs of the adduct. The ground-state binding of t he next correct nucleotide to the enzyme DNA complex was weakened by the ad duct 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 th ree strong pause sites was significantly weaker and less selective.