Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components

TY - JOUR

T1 - Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components

AU - Schomacher, Lars

AU - Schuerer, K. Anke

AU - Ciirdaeva, Elena

AU - McDermott, Paul

AU - Chong, James P. J.

AU - Kramer, Wilfried

AU - Fritz, Hans-Joachim

PY - 2010/4/4

Y1 - 2010/4/4

N2 - 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 Delta H. 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 Delta H. 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. (C) 2010 Elsevier B.V. All rights reserved.

AB - 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 Delta H. 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 Delta H. 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. (C) 2010 Elsevier B.V. All rights reserved.

KW - Hydrolytic deamination

KW - DNA uracil repair

KW - Strand incision repair

KW - DNA uridine endonuclease

KW - Thermophilic archaea

KW - Methanothermobacter thermautotrophicus

KW - Delta H

KW - THERMOAUTOTROPHICUM DELTA-H

KW - BASE EXCISION-REPAIR

KW - EXOIII HOMOLOG MTH212

KW - METHANOBACTERIUM-THERMOAUTOTROPHICUM

KW - INORGANIC PYROPHOSPHATASE

KW - COMPLETE GENOME

KW - POLYMERASE

KW - PROTEINS

KW - GLYCOSYLASE

KW - REPLICATION

UR - http://www.scopus.com/inward/record.url?scp=77949656067&partnerID=8YFLogxK

U2 - 10.1016/j.dnarep.2010.01.004

DO - 10.1016/j.dnarep.2010.01.004

M3 - Article

SN - 0921-8777

VL - 9

SP - 438

EP - 447

JO - Mutation research-Dna repair

JF - Mutation research-Dna repair

IS - 4

ER -