Team:Alberta/References/Publications/Structural basis for uracil recognition by archaeal family B DNA polymerases

From 2009.igem.org

Mark J. Fogg, Laurence H. Pearl & Bernard A. Connolly

Nature Structural Biology 9, 922 - 927 (2002)


Abstract: Deamination of cytosine to uracil in a G-C base pair is a major promutagenic event, generating G-CA-T mutations if not repaired before DNA replication. Archaeal family B DNA polymerases are uniquely able to recognize unrepaired uracil in a template strand and stall polymerization upstream of the lesion, thereby preventing the irreversible fixation of an A-T mutation. We have now identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provide this ability. The structure of this pocket provides interacting groups that discriminate uracil from the four normal DNA bases (including thymine). These groups are conserved in archaeal polymerases but absent from homologous viral polymerases that are unable to recognize uracil. Using site-directed mutagenesis, we have confirmed the biological role of this pocket and have engineered specific mutations in the Pfu polymerase that confer the ability to read through template-strand uracils and carry out PCR with dUTP in place of dTTP.


Link: [http://www.nature.com/nsmb/journal/v9/n12/full/nsb867.html Nature]

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