Increased catalytic activity and altered fidelity of human DNA polymerase iota in the presence of manganese.
All DNA polymerases require a divalent cation for catalytic activity. It is generally assumed that Mg(2+) is the physiological cofactor for replicative DNA polymerases in vivo. However, recent studies suggest that certain repair polymerases, such as pol lambda, may preferentially utilize Mn(2+) in vitro. Here we report on the effects of Mn(2+) and Mg(2+) on the enzymatic properties of human DNA polymerase iota (pol iota). pol iota exhibited the greatest activity in the presence of low levels of Mn(2+) (0.05-0.25 mm). Peak activity in the presence of Mg(2+) was observed in the range of 0.1-0.5 mm and was significantly reduced at concentrations >2 mm. Steady-state kinetic analyses revealed that Mn(2+) increases the catalytic activity of pol iota by approximately 30-60,000-fold through a dramatic decrease in the K(m) value for nucleotide incorporation. Interestingly, whereas pol iota preferentially misinserts G opposite T by a factor of approximately 1.4-2.5-fold over the correct base A in the presence of 0.25 and 5 mm Mg(2+), respectively, the correct insertion of A is actually favored 2-fold over the misincorporation of G in the presence of 0.075 mm Mn(2+). Low levels of Mn(2+) also dramatically increased the ability of pol iota to traverse a variety of DNA lesions in vitro. Titration experiments revealed a strong preference of pol iota for Mn(2+) even when Mg(2+) is present in a >10-fold excess. Our observations therefore raise the intriguing possibility that the cation utilized by pol iota in vivo may actually be Mn(2+) rather than Mg(2+), as tacitly assumed.
Frank
Ekaterina G
EG
Laboratory of Genomic Integrity, NICHD, National Institutes of Health, Bethesda, Maryland 20892-2725, USA.
Woodgate
Roger
R
D002384:Catalysis; D002412:Cations; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry / Q000494:pharmacology*; D008670:Metals / Q000737:chemistry; D008954:Models, Biological; D009711:Nucleotides / Q000737:chemistry; D000094769:DNA Polymerase iota
D002412:Cations; D017931:DNA Primers; D007477:Ions; D008670:Metals; D009711:Nucleotides; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; D000094769:DNA Polymerase iota; C000722070:POLI protein, human