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Hyperthermophilic Aquifex Aeolicus Initiates Primer Synthesis on a Limited Set of Trinucleotides Comprised of Cytosines and Guanines Marilynn A

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Hyperthermophilic Aquifex Aeolicus Initiates Primer Synthesis on a Limited Set of Trinucleotides Comprised of Cytosines and Guanines Marilynn A 5260–5269 Nucleic Acids Research, 2008, Vol. 36, No. 16 Published online 6 August 2008 doi:10.1093/nar/gkn461 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 Sayood3, 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 Downloaded from Received May 23, 2008; Revised June 27, 2008; Accepted July 2, 2008 ABSTRACT characterized although structural and functional adapta- tions have been described. The tRNAs from hyperthermo- http://nar.oxfordjournals.org/ The placement of the extreme thermophile Aquifex philes have extensive hydrogen bonding and numerous aeolicus in the bacterial phylogenetic tree has base modifications that restrict bending at crucial loca- evoked much controversy. We investigated whether tions, thereby increasing biomolecular thermostability adaptations for growth at high temperatures would (1). Chaperone proteins in the thermophilic bacterium alter a key functional component of the replication Thermus thermophilus have been shown to assist in folding machinery, specifically DnaG primase. Although or maintenance of structure (2). However, little effort has the structure of bacterial primases is conserved, been directed toward investigating adaptations of the DNA replication machinery in extreme thermophiles. the trinucleotide initiation specificity for A. aeolicus at University of Hawaii - Manoa on June 9, 2015 was hypothesized to differ from other microbes as Most hyperthermophiles belong to the domain an adaptation to a geothermal milieu. To determine Archaea, with the exception of a few bacteria (3). There- fore, the primase from the hyperthermophilic bacterium the full range of A. aeolicus primase activity, two Aquifex aeolicus was chosen to study the potential oligonucleotides were designed that comprised all mechanisms of thermoadaptation during DNA replication potential trinucleotide initiation sequences. One for comparison to other microbes. Structural analyses of of the screening templates supported primer synth- the bacterial primases studied to date show that these esis and the lengths of the resulting primers were enzymes are remarkably similar with three conserved used to predict possible initiation trinucleotides. functional domains that includes the zinc binding, RNA Use of trinucleotide-specific templates demon- polymerase and C-terminal domains (4). Primase, a spe- strated that the preferred initiation trinucleotide cialized DNA-dependent RNA polymerase, generates sequence for A. aeolicus primase was 5’-d(CCC)-3’. short oligoribonucleotide polymers de novo at the replica- Two other sequences, 5’-d(GCC)-3’ and d(CGC)-3’, tion fork (4). All known DNA polymerases are unable to initiate polymerization of nucleotides and require a free were also capable of supporting initiation, but to 0 a much lesser degree. None of these trinucleotides 3 -hydroxyl group for elongation. During DNA replica- tion, primase provides this essential 30-hydroxyl to initiate were known to be recognition sequences used by leading strand synthesis at least once and lagging strand other microbial primases. These results suggest synthesis numerous times. Conditional lethal mutations that the initiation specificity of A. aeolicus primase in the primase gene from Escherichia coli, Bacillus subtilis may represent an adaptation to a thermophilic and Staphylococcus aureus demonstrate the essential environment. nature of this enzyme (5À7). The Aquifex lineage is composed of nonsporeforming, hydrogen-oxidizing, obligate chemolithotrophic, micro- aerophilic, Gram-negative, rod-shaped bacteria. Aquifex INTRODUCTION aeolicus is one of the few bacteria known to be capable Molecular strategies in bacteria for adaptation to growth of survival at extremely high temperatures with a growth and survival at elevated temperatures have not been fully temperature maxima near 958C and an optimum growth *To whom correspondence should be addressed. Tel: 402 472 3429; Fax: 402 472 9402; Email: [email protected] ß 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. Nucleic Acids Research, 2008, Vol. 36, No. 16 5261 temperature of 858C (8). Interestingly, the genomes of Although the contiguous structure of A. aeolicus pri- these hyperthermophiles are only one-third the size of mase zinc-binding and RNA polymerase domains have the Gram-negative E. coli genome and demonstrate few been resolved by crystallography (26), the activity and specific indications of heat-resistance (9). Despite the the initiation specificity of this protein had not been intuitive expectation that thermophilic adaptations previously characterized. The lack of prior biochemical would occur, such as a higher G+C genome content and biological studies on this enzyme and the potential with a higher melting temperature (10À12), the A. aeolicus difference in trinucleotide specificity presented a chal- chromosome has a G+C content of only 43% (8). lenge for screening. Therefore, a new approach was devel- Therefore, there appears to be little relationship between oped with the capability to not only determine the activity adaptation of the organism to high growth temperatures of A. aeolicus primase, but also any other microbial pri- and high G+C content in the genome (9,13). mase that had not been characterized. To test as many Inferences made from the phylogenetic analysis of the trinucleotide sequences as possible, two oligonucleotides 16S ribosomal RNA sequence from A. aeolicus indicate were created using a genetic algorithm program such that this microbe is one of the earliest bacteria to have that all 64 trinucleotides were present in one or the diverged from the evolutionary tree of the eubacteria other sequence. After the resulting primer lengths were (14), but other studies suggest that RNA sequence changes determined, the potential initiation sequences were pre- Downloaded from were an adaptive response to the elevated temperature of dicted from the template and then confirmed using the organism’s environment (9,15). Protein phylogenies trinucleotide-specific templates. Using this approach, have placed A. aeolicus much further from the root, but the zinc binding and RNA polymerase domains of some studies could not identify a consistent node (8,16). A. aeolicus primase were shown to be sufficient to synthe- More recent cladistic studies using multiple signature pro- size primers de novo. Further, this replicative enzyme http://nar.oxfordjournals.org/ teins indicate that the Aquifex lineage emerged from initiated RNA polymer synthesis on trinucleotides not Gram-positive bacteria, prior to the split of Gram-positive previously known to be recognition sequences for any and Gram-negative bacteria (17,18). The proximity of microbial primase. Aquifex aeolicus primase preferen- Aquifex to Gram-positive bacteria is consistent with the tially initiated primer synthesis on 50-d(CCC)-30 and to low GC content of its genome and the encoding of both a lesser degree on 50-d(GCC)-30 and 50-d(CGC)-30. PolC and DnaE, replicative DNA polymerase III holoen- Elucidation of the initiation trinucleotides for A. aeolicus zymes. In contrast, Aquifex possesses a double membrane primase from this hyperthermophile has provided the structure similar to Gram-negative bacteria. Therefore, basis for further enzymatic studies. questions remain as to whether heat tolerance in A. aeolicus at University of Hawaii - Manoa on June 9, 2015 is a legacy of primordial bacterial evolution or a later adaptation to fill a vacant niche in the geothermal envir- MATERIALS AND METHODS onment. We hypothesized that a bacterial hyperthermo- Proteins, reagents and substrates phile might utilize primase initiation sequences that differ from mesophilic microbes. Residues 1À405 of A. aeolicus primase were cloned, Primases from several viruses, phages and bacteria stu- expressed and purified as previously described (26). The died to date have required a specific sequence to initiate C-terminal domain had been deleted to enhance protein synthesis of an RNA polymer de novo (4). The bacterio- solubility. The full-length primases from other bacteria, phages T4, T7 and SP6 recognize the trinucleotides including S. aureus and E. coli, were used to evaluate 50-d(GTT)-30,50-d(GTC)-30 and 50-d(GCA)-30, respectively the functionality of the two oligonucleotides. Escherichia (19À21). In E. coli, the primer is most often initiated by coli DnaG was isolated as previously described (27) and the sequence pppApG in vivo (22) and in vitro studies the other bacterial primases were cloned into pET30 and determined that E. coli primase primed single-stranded expressed in E. coli BL21(DE3) according to the manufac- DNA (ssDNA) templates containing 50-d(CTG)-30 in the turer’s suggestions (Novagen EMD Biosciences, La Jolla, absence of the replicative
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