Nucleic Acids Research Advance Access published online on August 6, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn461
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Nucleic Acid Enzymes |
Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines
1Department of Microbiology and Pathology, University of Nebraska Medical Center, Omaha, NE 68198-6495, 2University of Nebraska Center for Biosecurity, Omaha, NE 68198-4080, 3Department of Electrical Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0511, 4University of California, Berkeley, CA 94720 and 5Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA
*To whom correspondence should be addressed. Tel: 402 472 3429; Fax: 402 472 9402; Email: mgriep1{at}unl.edu
Received May 23, 2008. Revised June 27, 2008. Accepted July 2, 2008.
The placement of the extreme thermophile Aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. We investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically DnaG primase. Although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for A. aeolicus was hypothesized to differ from other microbes as an adaptation to a geothermal milieu. To determine the full range of A. aeolicus primase activity, two oligonucleotides were designed that comprised all potential trinucleotide initiation sequences. One of the screening templates supported primer synthesis and the lengths of the resulting primers were used to predict possible initiation trinucleotides. Use of trinucleotide-specific templates demonstrated that the preferred initiation trinucleotide sequence for A. aeolicus primase was 5'-d(CCC)-3'. Two other sequences, 5'-d(GCC)-3' and d(CGC)-3', were also capable of supporting initiation, but to a much lesser degree. None of these trinucleotides were known to be recognition sequences used by other microbial primases. These results suggest that the initiation specificity of A. aeolicus primase may represent an adaptation to a thermophilic environment.