NurA , a novel 5′–3′ nuclease gene linked to rad50 and mre11 homologs of thermophilic Archaea

Prokaryotic genomes acquire and eliminate blocks of DNA sequence by lateral gene transfer and spontaneous deletion, respectively. The basic parameters of spontaneous deletion, which are expected to influence the course of genome evolution, have not been determined for any hyperthermophilic archaeon. We therefore screened a number of independent pyrimidine auxotrophs of Sulfolobus acidocaldarius for deletions and sequenced those detected. Deletions accounted for only 0.4% of spontaneous pyrE mutations, corresponding to a frequency of about 10 ؊8 per cell. Nucleotide sequence analysis of five independent deletions showed no significant association of the endpoints with short direct repeats, despite the fact that several such repeats occur within the pyrE gene and that duplication mutations in pyrE reverted at high frequencies. Endpoints of the spontaneous deletions did not coincide with short inverted repeats or potential stem-loop structures. No consensus sequence common to all the deletions could be identified, although two deletions showed the potential of being stabilized by octanucleotide sequences elsewhere in pyrE, and another pair of deletions shared an octanucleotide at their 3 ends. The unusually low frequency and low sequence dependence of spontaneous deletions in the S. acidocaldarius pyrE gene compared to other genetic systems could not be explained in terms of possible constraints imposed by the 5-fluoroorotate selection.Sequence analysis of entire genomes has made it clear that despite the gradual accumulation of nucleotide substitutions, much of the divergence among prokaryotic species occurs through acquisition and loss of blocks of DNA sequences. Although bacterial genomes carry significant amounts of sequences originating in other lineages, related species exhibit similar genome sizes (21, 22), implying a general balance of losses against gains. Notable exceptions to this size constancy occur in species that have adopted a parasitic life strategy; the genomes of these species are either small or rapidly shrinking, reflecting the elimination of many genes whose functions have become superfluous (1,6,24). The mechanistic basis of genome reduction appears to be a short-term capacity to delete DNA that exceeds the capacity to acquire it through lateral gene transfer. This kinetic bias in favor of deletion implies that the maintenance of genome size seen in most bacterial lineages reflects selection for function of the vast majority of genes in the genome (24). It also explains the genome features observed in most free-living prokaryotes, including compactness (i.e., high open reading frame [ORF] density) and a dearth of fulllength but nonfunctional ORFs (1, 24).Data from genomes of hyperthermophilic archaea suggest a similar interplay among DNA acquisition, DNA loss, and selection for function. For example, Pyrococcus species can acquire large blocks of DNA by lateral transfer (9), yet they have also maintained relatively constant genome sizes during speciation (35). However, these and other archaea...

Section: Discussionsupporting

confidence: 88%

Molecular Characteristics of Spontaneous Deletions in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius

Grogan

Hansen

2003

“…5=-3= ssDNA/dsDNA exonuclease/endonuclease activities have been reported for S. acidocaldarius NurA (SacNurA) and Sulfolobus tokodaii NurA (StoNurA) (15,56), while no nuclease activity is observed with SsoNurA or PfuNurA until HerA is added (13,14,43). To identify the biochemical functions of DrNurA, we carried out a series of nuclease activity assays.…”

Section: Resultsmentioning

confidence: 99%

“…nurA and herA are usually located in operons. The nurA gene encodes a 5=-to-3= ssDNA/dsDNA exonuclease/endonuclease (15), and herA encodes an ATP-dependent bipolar helicase that unwinds dsDNA in both the 5=-to-3= and 3=-to-5= directions (16,17). Usually, mre11-rad50 colocalizes with this operon (18), and nurA, herA, mre11, and rad50 are cotranscribed in response to UV irradiation (19,20).…”

mentioning

confidence: 99%

Biochemical and Functional Characterization of the NurA-HerA Complex from Deinococcus radiodurans

Cheng

Chen

et al. 2015

In archaea, the NurA nuclease and HerA ATPase/helicase, together with the Mre11-Rad50 complex, function in 3= singlestranded DNA (ssDNA) end processing during homologous recombination (HR). However, bacterial homologs of NurA and HerA have not been characterized. From Deinococcus radiodurans, we identified the manganese-dependent 5=-to-3= ssDNA/double-stranded DNA (dsDNA) exonuclease/endonuclease NurA (DrNurA) and the ATPase HerA (DrHerA). These two proteins stimulated each other's activity through direct protein-protein interactions. The N-terminal HAS domain of DrHerA was the key domain for this interaction. Several critical residues of DrNurA and DrHerA were verified by site-directed mutational analysis. Temperature-dependent activity assays confirmed that the two proteins had mesophilic features, with optimum activity temperatures 10°C to 15°C higher than their optimum growth temperatures. Knocking out either nurA or herA affected cell proliferation by shortening the growth phase, especially for growth at a high temperature (37°C). In addition, both mutant strains displayed almost 10-fold-reduced intermolecular recombination efficiency, indicating that DrNurA and DrHerA might be involved in homologous recombination in vivo. However, single-and double-gene deletions did not show significantly decreased radioresistance. Our results confirmed that the biochemical activities of bacterial NurA and HerA proteins were conserved with archaea. Our phenotypical results suggested that these proteins might have different functions in bacteria. IMPORTANCEDeinococcus radiodurans NurA (DrNurA) was identified as a manganese-dependent 5=-to-3= ssDNA/dsDNA exonuclease/endonuclease, and Deinococcus radiodurans HerA (DrHerA) was identified as an ATPase. Physical interactions between DrNurA and DrHerA explained mutual stimulation of their activities. The N-terminal HAS domain on DrHerA was identified as the interaction domain. Several essential functional sites on DrNurA and DrHerA were characterized. Both DrHerA and DrNurA showed mesophilic biochemical features, with their optimum activity temperatures 10°C to 15°C higher than their optimum growth temperatures in vitro. Knockout of nurA or herA led to abnormal cell proliferation and reduced intermolecular recombination efficiency but no obvious effect on radioresistence.

“…Transformants were grown overnight at 37°C and used to inoculate 1-liter cultures that were incubated to an A 600 of approximately 0.8. The cultures were then placed on ice to induce cold shock proteins which may prevent inclusion body formation (7). After 1 h, recombinant protein expression was induced by adding 0.5 mM IPTG (isopropyl-␤-D-thiogalactopyranoside).…”

Section: Vol 188 2006 a Novel ␣-Galactosidase From S Solfataricus mentioning

confidence: 99%

Identification of a Novel α-Galactosidase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

Brouns

Smits

et al. 2006

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable ␣-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for ␣-linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable ␣-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.␣-Galactosidases (␣-Gals) (EC 3.2.1.22) are a widespread class of enzymes that liberate galactose from the nonreducing end of sugars. In bacteria, yeasts, and fungi, these enzymes are usually involved in the degradation of various plant saccharides, which can then serve as a carbon and energy source for growth. Plants synthesize ␣-galactosides such as raffinose and stachyose as the major energy storage molecules in leaves, roots, and tubers. Moreover, these oligosaccharides have been associated with cold and desiccation tolerance of seeds (28). Both raffinose and stachyose are produced by two specialized synthases (raffinose synthase [EC 2.4.1.82] and stachyose synthase [EC 2.4.1.67]) that use galactinol as a galactosyl donor (39). The oligosaccharides are degraded during seed germination by the action of two distinct types of ␣-Gals that differ in their optimal pH of catalysis. While the acid ␣-Gal type is most likely active in the acidic environment of the vacuole and the apoplasm, the alkaline ␣-Gal type probably catalyzes galactose release in the more neutral or alkaline cytoplasm (4,17,31). Mammals express an ␣-Gal and an ␣-N-acetylgalactosaminidase (␣-NAGal) in lysosomal bodies to degrade glycolipids, glycoproteins, and oligosaccharides. In humans, mutations in