Trapping of DNA topoisomerase I on nick-containing DNA in cell free extracts of Saccharomyces cerevisiae.
The aim of the present study was to identify proteins that bind nicked DNA intermediates formed in the course of base excision repair (BER) in cell free extracts of Saccharomyces cerevisiae. In mammalian cells, nicks in DNA are targets of proteins such as PARP-1 or XRCC1 that have no homologues in yeast. One of the most promising methodologies to trap proteins that interact with damaged DNA lies in using a photocrosslinking technique with photoactivable dNTP analogues such as exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) for enzymatic synthesis of DNA probes with a photoreactive dNMP residue at the 3'-margin of a nick. Using this approach, we identified a major covalent DNA-protein adduct between a nick-containing 34-mer DNA duplex and a protein of a molecular mass of around 100-kDa. Unexpectedly, the formation of the 100-kDa adduct did not require the incorporation of the photoreactive dNMP residue at the 3'-margin of the nick nor exposure to near UV-light. However, the formation of the 100-kDa adduct strictly required a nick or a short gap in the DNA probe. Furthermore, the 100-kDa adduct was not detected in yeast extracts lacking DNA topoisomerase I (Top1). To further establish the nature of crosslinked protein, yeast Top1 was tagged with a Myc-epitope. In this case, the mobility of the Top1-DNA adduct increased by 7- kDa. Therefore, our data speak in favor of Top1 trapping by nicked DNA. In support of this hypothesis, purified yeast Top1 was also crosslinked to nicked DNA structures. Undamaged, uracil- and abasic (AP) site-containing DNAs were unable to trap Top1 under the same assay conditions. Since nicked DNA structures are frequently formed in the course of BER, their covalent linkage to Top1 has the potential to interfere with BER in vivo.
Lebedeva
Natalia
N
CEA, UMR217 CNRS Radiobiologie Moléculaire et Cellulaire, route du Panorama, BP6, 92265-Fontenay aux Roses, France.
Auffret Vander Kemp
Patricia
P
Bjornsti
Mary-Ann
MA
Lavrik
Olga
O
Boiteux
Serge
S
D000345:Affinity Labels; D001483:Base Sequence; D002474:Cell-Free System; D003432:Cross-Linking Reagents; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta; D004264:DNA Topoisomerases, Type I