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1.  Endogenous Mutagenesis in Recombinant Sulfolobus Plasmids 
Journal of Bacteriology  2013;195(12):2776-2785.
Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-d-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac− mutants also grew faster than the Lac+ parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10−4 mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G·C-to-A·T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the “transcription-associated mutagenesis” seen in bacteria and eukaryotes.
PMCID: PMC3697259  PMID: 23564176
2.  Sulfolobus Mutants, Generated via PCR Products, Which Lack Putative Enzymes of UV Photoproduct Repair 
Archaea  2011;2011:864015.
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.
PMCID: PMC3139894  PMID: 21785574
3.  Break-Induced Replication and Genome Stability 
Biomolecules  2012;2(4):483-504.
Genetic instabilities, including mutations and chromosomal rearrangements, lead to cancer and other diseases in humans and play an important role in evolution. A frequent cause of genetic instabilities is double-strand DNA breaks (DSBs), which may arise from a wide range of exogeneous and endogeneous cellular factors. Although the repair of DSBs is required, some repair pathways are dangerous because they may destabilize the genome. One such pathway, break-induced replication (BIR), is the mechanism for repairing DSBs that possesses only one repairable end. This situation commonly arises as a result of eroded telomeres or collapsed replication forks. Although BIR plays a positive role in repairing DSBs, it can alternatively be a dangerous source of several types of genetic instabilities, including loss of heterozygosity, telomere maintenance in the absence of telomerase, and non-reciprocal translocations. Also, mutation rates in BIR are about 1000 times higher as compared to normal DNA replication. In addition, micro-homology-mediated BIR (MMBIR), which is a mechanism related to BIR, can generate copy-number variations (CNVs) as well as various complex chromosomal rearrangements. Overall, activation of BIR may contribute to genomic destabilization resulting in substantial biological consequences including those affecting human health.
PMCID: PMC3678771  PMID: 23767011
double-strand break (DSB); DNA repair; break-induced replication (BIR); recombination
4.  Roles of the Y-family DNA Polymerase Dbh in Accurate Replication of the Sulfolobus Genome at Extremely High Temperature 
DNA repair  2012;11(4):391-400.
The intrinsically thermostable Y-family DNA polymerases of Sulfolobus spp. have revealed detailed three-dimensional structure and catalytic mechanisms of trans-lesion DNA polymerases, yet their functions in maintaining their native genomes remain largely unexplored. To identify functions of the Y-family DNA polymerase Dbh in replicating the Sulfolobus genome under extreme conditions, we disrupted the dbh gene in Sulfolobus acidocaldarius and characterized the resulting mutant strains phenotypically. Disruption of dbh did not cause any obvious growth defect, sensitivity to any of several DNA-damaging agents, or change in overall rate of spontaneous mutation at a well-characterized target gene. Loss of dbh did, however, cause significant changes in the spectrum of spontaneous forward mutation in each of two orthologous target genes of different sequence. Relative to wild-type strains, dbh− constructs exhibited fewer frame-shift and other small insertion-deletion mutations, but exhibited more base-pair substitutions that converted G:C base pairs to T:A base pairs. These changes, which were confirmed to be statistically significant, indicate two distinct activities of the Dbh polymerase in Sulfolobus cells growing under nearly optimal culture conditions (78-80 °C and pH 3). The first activity promotes slipped-strand events within simple repetitive motifs, such as mononucleotide runs or triplet repeats, and the second promotes insertion of C opposite a potentially miscoding form of G, thereby avoiding G:C to T:A transversions.
PMCID: PMC3591481  PMID: 22305938
Trans-lesion DNA synthesis; Y-family DNA polymerase; Sulfolobus DNA polymerase Dbh; DNA-damage sensitivity; Spontaneous mutation spectra

Results 1-4 (4)