Sulfolobus mutants, generated via PCR products, which lack putative enzymes of UV photoproduct repair.

Cynthia J Sakofsky,Dennis W Grogan,Laura A Runck

doi:10.1155/2011/864015

Abstract

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.

Highlights

Gene inventories of hyperthermophilic archaea (HA) suggest that this deeply branching clade of prokaryotes may employ unusual molecular strategies during DNA replication and repair
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 lack the UvrABC homologues that mediate nucleotide excision repair (NER) in bacteria and mesophilic archaea [5]
The resulting plasmid is transformed into a pyrE recipient, and the selected gene integrates into the Sulfolobus chromosome through homologous recombination [14,15,16]. We have found this approach to be effective for genes of Sulfolobus acidocaldarius, published studies show that the recombination system of S. acidocaldarius can operate on much shorter DNA sequences, that is, down to 30 bp or less [17,18,19]

Summary

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. 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 ahead of dU residues in the template strand [2, 3]. 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 lack the UvrABC homologues that mediate nucleotide excision repair (NER) in bacteria and mesophilic archaea [5]. 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

Methods

Results

Conclusion