Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components.
Hydrolytic deamination of DNA cytosine residues results in U/G mispairs, pre-mutagenic lesions threatening long-term genetic stability. Hence, DNA uracil repair is ubiquitous throughout all extant life forms and base excision repair, triggered by a uracil DNA glycosylase (UDG), is the mechanistic paradigm adopted, as it seems, by all bacteria and eukaryotes and a large fraction of archaea. However, members of the UDG superfamily of enzymes are absent from the extremely thermophilic archaeon Methanothermobacter thermautotrophicus DeltaH. This organism, as a hitherto unique case, initiates repair by direct strand incision next to the DNA-U residue, a reaction catalyzed by the DNA uridine endonuclease Mth212, an ExoIII homologue. To elucidate the detailed mechanism, in particular to identify the molecular partners contributing to this repair process, we reconstituted DNA uracil repair in vitro from only four purified enzymes of M. thermautotrophicus DeltaH. After incision at the 5'-side of a 2'-d-uridine residue by Mth212 DNA polymerase B (mthPolB) is able to take over the 3'-OH terminus and carry out repair synthesis generating a 5'-flap structure that is resolved by mthFEN, a 5'-flap endonuclease. Finally, DNA ligase seals the resulting nick. This defines mechanism and minimal enzymatic requirements of DNA-U repair in this organism.
Schomacher
Lars
L
Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Germany.
Schürer
K Anke
KA
Ciirdaeva
Elena
E
McDermott
Paul
P
Chong
James P J
JP
Kramer
Wilfried
W
Fritz
Hans-Joachim
HJ
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D019641:DNA, Archaeal / Q000378:metabolism*; D017013:Methanobacteriaceae / Q000378:metabolism*; D008954:Models, Biological; D014498:Uracil / Q000378:metabolism*; D051981:Uracil-DNA Glycosidase / Q000378:metabolism
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D014498:Uracil; D051981:Uracil-DNA Glycosidase; D011088:DNA Ligases; D000072481:DNA Ligase ATP