5260–5269 Nucleic Acids Research, 2008, Vol. 36, No. 16 doi:10.1093/nar/gkn461 Published online 6 August 2008 Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines Marilynn A. Larson1,2, Rafael Bressani1,2, Khalid Sayood 3 , Jacob E. Corn4, James M. Berger4, Mark A. Griep5,* and Steven H. Hinrichs1,2 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 Received May 23, 2008; Revised June 27, 2008; Accepted July 2, 2008 ABSTRACT 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 synth- esis and the lengths of the resulting primers were used to predict possible initiation trinucleotides. Use of trinucleotide-specific templates demon- strated 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. INTRODUCTION Molecular strategies in bacteria for adaptation to growth and survival at elevated temperatures have not been fully characterized although structural and functional adapta- tions have been described. The tRNAs from hyperthermo- philes have extensive hydrogen bonding and numerous base modiﬁcations that restrict bending at crucial loca- tions, thereby increasing biomolecular thermostability (1). Chaperone proteins in the thermophilic bacterium Thermus thermophilus have been shown to assist in folding or maintenance of structure (2). However, little eﬀort has been directed toward investigating adaptations of the DNA replication machinery in extreme thermophiles. Most hyperthermophiles belong to the domain Archaea, with the exception of a few bacteria (3). There- fore, the primase from the hyperthermophilic bacterium Aquifex aeolicus was chosen to study the potential mechanisms of thermoadaptation during DNA replication for comparison to other microbes. Structural analyses of the bacterial primases studied to date show that these enzymes are remarkably similar with three conserved functional domains that includes the zinc binding, RNA polymerase and C-terminal domains (4). Primase, a spe- cialized DNA-dependent RNA polymerase, generates short oligoribonucleotide polymers de novo at the replica- tion fork (4). All known DNA polymerases are unable to initiate polymerization of nucleotides and require a free 30-hydroxyl group for elongation. During DNA replica- tion, primase provides this essential 30-hydroxyl to initiate leading strand synthesis at least once and lagging strand synthesis numerous times. Conditional lethal mutations in the primase gene from Escherichia coli, Bacillus subtilis and Staphylococcus aureus demonstrate the essential nature of this enzyme (5 7). The Aquifex lineage is composed of nonsporeforming, hydrogen-oxidizing, obligate chemolithotrophic, micro- aerophilic, Gram-negative, rod-shaped bacteria. Aquifex aeolicus is one of the few bacteria known to be capable of survival at extremely high temperatures with a growth temperature maxima near 958C and an optimum growth *To whom correspondence should be addressed. Tel: 402 472 3429; Fax: 402 472 9402; Email: mgriep1@unl.edu 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.