In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096) were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5′ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains. 1. Introduction Gene inventories of hyperthermophilic archaea (HA) suggest that this deeply branching clade of prokaryotes may employ unusual molecular strategies during DNA replication and repair. For example, HA have certain DNA enzymes and enzymatic properties not found in mesophilic archaea or bacteria. These include reverse DNA gyrase, a type I topoisomerase that introduces positive superhelical turns into DNA [1], and family-B DNA polymerases which stall specifically ahead of dU residues in the template strand [2, 3]. Conversely, HA lack certain DNA-repair proteins that are widely conserved in other organisms; they encode no homologues of DNA mismatch repair proteins MutS and MutL [4], and they also lack the UvrABC homologues that mediate nucleotide excision repair (NER) in bacteria and mesophilic archaea [5]. Whereas HA do encode homologues of the eukaryotic helicases and structure-specific nucleases that complete the process of NER in eukaryotes, they do not have the corresponding proteins that initiate NER. The fact that no HA encode homologues of known NER-specific damage-recognition proteins seems significant, since these proteins are required for NER function in other organisms, and are the only proteins specific to NER in eukaryotes. Experimental data, nevertheless, suggest that HA can excise UV photoproducts from DNA although the mechanism and proteins responsible have not been identified. Biochemical assays indicate time-dependent loss of cyclobutane pyrimidine dimers from genomic DNA of intact Sulfolobus solfataricus cells at physiological temperature [6, 7]. In Sulfolobus acidocaldarius conjugation assays, UV enhances recombinant formation,
References
[1]
P. Forterre, “A hot story from comparative genomics: reverse gyrase is the only hyperthermophile-specific protein,” Trends in Genetics, vol. 18, no. 5, pp. 236–238, 2002.
[2]
R. S. Lasken, D. M. Schuster, and A. Rashtchian, “Archaebacterial DNA polymerases tightly bind uracil-containing DNA,” Journal of Biological Chemistry, vol. 271, no. 30, pp. 17692–17696, 1996.
[3]
G. Shuttleworth, M. J. Fogg, M. R. Kurpiewski, L. Jen-Jacobson, and B. A. Connolly, “Recognition of the pro-mutagenic base uracil by family B DNA polymerases from archaea,” Journal of Molecular Biology, vol. 337, no. 3, pp. 621–634, 2004.
[4]
P. Sachadyn, “Conservation and diversity of MutS proteins,” Mutation Research, vol. 694, no. 1-2, pp. 20–30, 2010.
[5]
M. F. White and D. W. Grogan, “DNA stability and repair,” in Thermophiles: Biology and Technology at High Temperatures, F. T. Robb, G. Antranikian, G. Antranikian, D. W. Grogan, and A. J. Driessen, Eds., pp. 179–187, CRC Press, Boca Raton, Fla, USA, 2008.
[6]
R. Dorazi, D. G？tz, S. Munro, R. Bernander, and M. F. White, “Equal rates of repair of DNA photoproducts in transcribed and non-transcribed strands in Sulfolobus solfataricus,” Molecular Microbiology, vol. 63, no. 2, pp. 521–529, 2007.
[7]
V. Romano, A. Napoli, V. Salerno, A. Valenti, M. Rossi, and M. Ciaramella, “Lack of strand-specific repair of UV-induced DNA lesions in three genes of the Archaeon Sulfolobus solfataricus,” Journal of Molecular Biology, vol. 365, no. 4, pp. 921–929, 2007.
[8]
K. J. Schmidt, K. E. Beck, and D. W. Grogan, “UV stimulation of chromosomal marker exchange in Sulfolobus acidocaldarius: implications for DNA repair, conjugation and homologous recombination at extremely high temperatures,” Genetics, vol. 152, no. 4, pp. 1407–1415, 1999.
[9]
M. Fujihashi, N. Numoto, Y. Kobayashi et al., “Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor,” Journal of Molecular Biology, vol. 365, no. 4, pp. 903–910, 2007.
[10]
D. W. Grogan, “Photoreactivation in an archaeon from geothermal environments,” Microbiology, vol. 143, no. 4, pp. 1071–1076, 1997.
[11]
R. Yonemasu, S. J. McCready, J. M. Murray et al., “Characterization of the alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe,” Nucleic Acids Research, vol. 25, no. 8, pp. 1553–1558, 1997.
[12]
A. M. Earl, S. K. Rankin, K. P. Kim, O. N. Lamendola, and J. R. Battista, “Genetic evidence that the uvsE gene product of Deinococcus radiodurans R1 is a UV damage endonuclease,” Journal of Bacteriology, vol. 184, no. 4, pp. 1003–1009, 2002.
[13]
Z. Sancar, K. A. Franklin, and G. B. Sancar, “Escherichia coli DNA photolyase stimulates uvrABC excision nuclease in vitro,” Proceedings of the National Academy of Sciences of the United States of America, vol. 81, no. 23 I, pp. 7397–7401, 1984.
[14]
P. Worthington, V. Hoang, F. Perez-Pomares, and P. Blum, “Targeted disruption of the α-amylase gene in the hyperthermophilic archaeon Sulfolobus solfataricus,” Journal of Bacteriology, vol. 185, no. 2, pp. 482–488, 2003.
[15]
S. V. Albers and A. J. M. Driessen, “Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome,” Archaea, vol. 2, no. 3, pp. 145–149, 2008.
[16]
L. Deng, H. Zhu, Z. Chen, Y. X. Liang, and Q. She, “Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus,” Extremophiles, vol. 13, no. 4, pp. 735–746, 2009.
[17]
J. E. Hansen, A. C. Dill, and D. W. Grogan, “Conjugational genetic exchange in the hyperthermophilic archaeon Sulfolobus acidocaldarius: intragenic recombination with minimal dependence on marker separation,” Journal of Bacteriology, vol. 187, no. 2, pp. 805–809, 2005.
[18]
N. Kurosawa and D. W. Grogan, “Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: properties and uses,” FEMS Microbiology Letters, vol. 253, no. 1, pp. 141–149, 2005.
[19]
D. W. Grogan and J. Rockwood, “Discontinuity and limited linkage in the homologous recombination system of a hyperthermophilic archaeon,” Journal of Bacteriology, vol. 192, no. 18, pp. 4660–4668, 2010.
[20]
D. E. Kelly, D. C. Lamb, and S. L. Kelly, “Genome-wide generation of yeast gene deletion strains,” Comparative and Functional Genomics, vol. 2, no. 4, pp. 236–242, 2001.
[21]
M. S. Reilly and D. W. Grogan, “Characterization of intragenic recombination in a hyperthermophilic archaeon via conjugational DNA exchange,” Journal of Bacteriology, vol. 183, no. 9, pp. 2943–2946, 2001.
[22]
D. W. Grogan and R. P. Gunsalus, “Sulfolobus acidocaldarius synthesizes UMP via a standard de novo pathway: results of a biochemical-genetic study,” Journal of Bacteriology, vol. 175, no. 5, pp. 1500–1507, 1993.
[23]
D. W. Grogan and K. R. Stengel, “Recombination of synthetic oligonucleotides with prokaryotic chromosomes: substrate requirements of the Escherichia coli/λrd and Sulfolobus acidocaldarius recombination systems,” Molecular Microbiology, vol. 69, no. 5, pp. 1255–1265, 2008.
[24]
M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, “A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector,” Molecular Microbiology, vol. 48, no. 5, pp. 1241–1252, 2003.
[25]
E. R. Wood, F. Ghané, and D. W. Grogan, “Genetic responses of the thermophilic archaeon Sulfolobus acidocaldarius to short-wavelength UV light,” Journal of Bacteriology, vol. 179, no. 18, pp. 5693–5698, 1997.
[26]
D. W. Grogan, G. T. Carver, and J. W. Drake, “Genetic fidelity under harsh conditions: analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 14, pp. 7928–7933, 2001.
[27]
D. W. Grogan, “Exchange of genetic markers at extremely high temperatures in the archaeon Sulfolobus acidocaldarius,” Journal of Bacteriology, vol. 178, no. 11, pp. 3207–3211, 1996.
[28]
QI. Zheng, “Statistical and algorithmic methods for fluctuation analysis with SALVADOR as an implementation,” Mathematical Biosciences, vol. 176, no. 2, pp. 237–252, 2002.
[29]
B. M. Hall, C. X. Ma, P. Liang, and K. K. Singh, “Fluctuation anaLysis calculator: a web tool for the determination of mutation rate using Luria-Delbück fluctuation analysis,” Bioinformatics, vol. 25, no. 12, pp. 1564–1565, 2009.
[30]
N. F. Cariello, W. W. Piegorsch, W. T. Adams, and T. R. Skopek, “Computer program for the analysis of mutational spectra application to p53 mutations,” Carcinogenesis, vol. 15, no. 10, pp. 2281–2285, 1994.
[31]
M. Tanaka, I. Narumi, T. Funayama et al., “Characterization of pathways dependent on the uvsE, uvrA1, or uvrA2 gene product for UV resistance in Deinococcus radiodurans,” Journal of Bacteriology, vol. 187, no. 11, pp. 3693–3697, 2005.
[32]
R. D. Finn, J. Mistry, J. Tate et al., “The Pfam protein families database,” Nucleic Acids Research, vol. 38, no. 1, pp. D211–D222, 2009.
[33]
T. Ueda, A. Kato, S. Kuramitsu, H. Terasawa, and I. Shimada, “Identification and characterization of a second chromophore of DNA photolyase from Thermus thermophilus HB27,” Journal of Biological Chemistry, vol. 280, no. 43, pp. 36237–36243, 2005.
[34]
M. E. Fox, B. J. Feldman, and G. Chu, “A novel role for DNA photolyase: binding to DNA damaged by drugs is associated with enhanced cytotoxicity in Saccharomyces cerevisiae,” Molecular and Cellular Biology, vol. 14, no. 12, pp. 8071–8077, 1994.
[35]
M. A. Pritchett, J. K. Zhang, and W. W. Metcalf, “Development of a markerless genetic exchange method for Methanosarcina acetivorans C2A and its use in construction of new genetic tools for methanogenic archaea,” Applied and Environmental Microbiology, vol. 70, no. 3, pp. 1425–1433, 2004.
