Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity.
We found that both RNA and cDNA preparations derived from melanocytes contain a RT-PCR inhibitor that copurified with nucleic acids. Investigation of the candidate inhibitor melanin revealed that it potently blocks PCR at concentrations below 200 ng/ml, whereas 100 microg/ml melanin was required to inhibit reverse transcription. Melanin and thermostable DNA polymerase preferentially formed a distinct complex with reduced migration velocity as compared to pure polymerase in nondenaturating polyacrylamide gel electrophoresis. The inhibition of the enzyme by melanin could be reversed by diluting solutions of preformed complexes or by adding excess amounts of other proteins such as bovine serum albumin or dry milk. Our findings demonstrate that melanin is a potent inhibitor of thermostable DNA polymerase in vitro and that the inhibitory effect is conferred by a direct and reversible polymerase-melanin interaction.
Eckhart
L
L
Department of Dermatology, University Vienna Medical School, Vienna, Austria.
Bach
J
J
Ban
J
J
Tschachler
E
E
D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008543:Melanins / Q000494:pharmacology*; D008544:Melanocytes / Q000201:enzymology*; D019384:Nucleic Acid Synthesis Inhibitors; D011485:Protein Binding; D012313:RNA / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D019914:Taq Polymerase / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D008543:Melanins; D019384:Nucleic Acid Synthesis Inhibitors; D012710:Serum Albumin, Bovine; D012313:RNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase