Genetic analysis of an archaeal Holliday junction resolvase in Escherichia coli.
The study of genes and proteins in heterologous model systems provides a powerful approach to the analysis of common processes in biology. Here, we show how the bacterium Escherichia coli can be exploited to analyse genetically and biochemically the activity and function of a Holliday junction resolving enzyme from an archaeal species. We have purified and characterised a member of the newly discovered Holliday junction cleaving (Hjc) family of resolvases from the moderately thermophilic archaeon Methanobacterium thermoautotrophicum and demonstrate that it promotes DNA repair in resolvase-deficient ruv mutants of E. coli. The data presented provide the first direct evidence that such archaeal enzymes can promote DNA repair in vivo, and support the view that formation and resolution of Holliday junctions are key to the interplay between DNA replication, recombination and repair in all organisms. We also show that Hjc promotes DNA repair in E. coli in a manner that requires the presence of the RecG branch migration protein. These results support models in which RecG acts at a replication fork stalled at a lesion in the DNA, catalysing fork regression and forming a Holliday junction that can then be acted upon by Hjc.
Bolt
E L
EL
Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham, NG7 2UH, UK.
Lloyd
R G
RG
Sharples
G J
GJ
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics / Q000528:radiation effects; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D029968:Escherichia coli Proteins; D045565:Holliday Junction Resolvases; D017014:Methanobacterium / Q000201:enzymology* / Q000235:genetics; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011995:Recombination, Genetic / Q000235:genetics*; D013379:Substrate Specificity; D014466:Ultraviolet Rays
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C071446:RecG protein, E coli; D004247:DNA; D004706:Endodeoxyribonucleases; D045565:Holliday Junction Resolvases; D004265:DNA Helicases