Mechanisms By Which DNA And RNA Polymerases Discriminate Between Right And Wrong (d)NTPs

• Published in: The FASEB journal 2006-03, Vol.20 (4), p.A512-A513
• Main Authors: Kuchta, Robert D, Beckman, Jeff, Kincaid, Kristi, Moore, Chad, Loi, Michele, Timblin, Greg
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.20.4.A512-d

A key question regarding DNA and RNA polymerase mechanisms is how they discriminate between right and wrong (d)NTPs. Even after extensive study, the answer(s) to this question remain unclear and controversial. We have addressed this question using specific series of modified (d)NTPs and 3 enzymes: Human DNA primase, an X family polymerase that exhibits very low fidelity (average error frequency of 1:100); Herpes primase, an extremely low fidelity polymerase (average error frequency of 1:30), and; human DNA polymerase α, a B family polymerase of moderately high fidelity (average error frequency of 1:5000). Both primases only efficiently polymerize a NTP if it can form Watson‐Crick base‐pairs with the template base being replicated. Importantly, these enzymes can accomplish this using either the major or minor tautomeric forms of the base. Pol α, in contrast, uses a fundamentally different mechanism to identify the incoming as either right or wrong. Rather than obtain fidelity by selecting for the correct dNTP, pol α primarily obtains fidelity by selecting against the 3 wrong dNTPs (i.e., negative selectivity). On a purine dNTP, pol α mainly uses N‐1 and the exocyclic group at C‐6 to identify the incoming dNTP as wrong. Together, these data indicate that polymerases have developed multiple solutions to discriminating between right and wrong (d)NTPs. This work was supported by grants from the NIH (AI59764) and the U.S. Army (W911NF‐05‐1‐0172).