Stalling of DNA replication in response to template strand Uracil by archaeal DNA polymerases
Deamination of Cytosine in DNA G:C base-pairs gives a pro-mutagenic G:U mismatch, which, on replication, results in a G:C → A:T transition mutation in 50% of the progeny. All organisms contain enzymes that safeguard the genome from the mutagenic effects of Uracil: Uracil-DNA-glycosylases, which excise uracil from DNA, and dUTPase which prevents de novo incorporation of Uracil into DNA. Our group has discovered a novel Uracil protection mechanism embedded in the DNA polymerase responsible for copying DNA in the archaea. The polymerase has a Uracil binding pocket that scans the DNA template ahead of the replication fork and stalls polymerisation when the base is encountered, preventing the irreversible copying of the G:U mismatch and fixation of the A:T mutation in half the progeny. The polymerase offers the last opportunity to deal with the detrimental effects of Uracil and replication stalling is the first example of a novel DNA pathway based on template strand proofreading. Such a mechanism might be especially appropriate to the archaea, a domain of life often associated with extreme environments such as the high temperatures expected to promote cytosine deamination. Archaeal DNA polymerases are commonly employed in the polymerase chain reaction (PCR), the most commonly used protocol in genetic engineering. The discovery of uracil-induced stalling has directly resulted in polymerases with superior properties in the PCR. The natural polymerase often delivers poor PCR performance due to “uracil-poisoning”; here traces of Uracil present in the DNA inhibit the polymerase, preventing DNA amplification. Polymerase variants that are insensitive to uracil were obtained to give far improved performance in the PCR. These mutants were patented and licensed to a biotechnology firm. They are now commercially available and generating considerable Royalties for the University.
Fogg MJ, Pearl LH and Connolly BA. 2002. Structural basis for uracil recognition by archaeal family B DNA polymerases. Nature Structural Biology 9, 922-927.
Shuttleworth G, Fogg MJ, Kurpiewski MR, Jen-Jacobson L and Connolly BA. 2004. Recognition of the pro-mutageneic base uracil by family B DNA polymerases from archaea. Journal of Molecular Biology 337, 621-634.
