The effect of carcinogenic adducts on in vitro DNA replication by a reconstituted system

Bacteriophage T7 gene 4 protein is an essential component of the T7 DNA replication system. Gene 4 protein acts as a helicase and primase to facilitate simultaneous synthesis on both the leading and lagging strands at the replication fork. The binding of gene 4 protein to single-stranded DNA requires an NTP cofactor, preferably dTTP in vitro. Measuring the rate and extent of TDP formation is a means of directly measuring the rate and extent of gene 4 protein translocation.;We have measured the rate and extent of hydrolysis of TTP by the gene 4 protein on native and benzo(a) pyrene (B(a) P)-modified DNA and find that these bulky adducts pose a strong impediment to the movement of the gene 4 protein. Time course experiments on adduct-containing DNA show that after an initial burst of hydrolysis, which parallels what is observed on unmodified DNA, that further hydrolysis abruptly ceases. Addition of excess unmodified DNA does not restore the hydrolysis activity suggesting that the gene 4 protein is being sequestered on the DNA at the site of the adduct. This was confirmed by digestion/protection experiments in which gene 4 protein preferentially protected the radiolabeled adducts as opposed to randomly labelled M13 DNA. By isolating the gene 4 protein-DNA complex and identifying the bound cofactor, we were able to propose a new model for gene 4 protein translocation.;We have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo(a) pyrene adducts on DNA synthesis using native and modified single- and double-stranded M13 DNA templates. These adducts act as blocks to synthesis by this system and overall synthesis decreases as the level of adducts per molecule of DNA increases. Gene 4 protein-primed reactions were inhibited to a greater extent than oligonucleotide-primed reactions. We also found that T7 DNA polymerase freely dissociates after encountering an adduct during oligonucleotide primed synthesis reactions. The gene 4 protein does not dissociate unless T7 DNA polymerase is present, suggesting a physical association between these proteins during synthesis. The ratio of hydrolysis to synthesis is maintained on DNA containing various levels of B(a) P adducts as is the ratio of hydrolysis which occurs in the presence or absence of DNA synthesis, suggesting that a major portion of the inhibition of synthesis observed in this system is the blockage of the translocation of the gene 4 protein.;Finally, we found evidence that gene 4 protein helicase activity or the polymerase-gene 4 protein complex is inhibited by adducts in the displaced strand during synthesis on strand-specifically modified double-stranded templates.