End modification of a linear DNA duplex enhances NER-mediated excision of an internal Pt(II)-lesion.
The study of DNA repair has been facilitated by the development of extract-based in vitro assay systems and the use of synthetic DNA duplexes that contain site-specific lesions as repair substrates. Unfortunately, exposed DNA termini can be a liability when working in crude cell extracts because they are targets for DNA end-modifying enzymes and binding sites for proteins that recognize DNA termini. In particular, the double-strand break repair protein Ku is an abundant DNA end-binding protein that has been shown to interfere with nucleotide excision repair (NER) in vitro. To facilitate the investigation of NER in whole-cell extracts, we explored ways of modifying the exposed ends of synthetic repair substrates to prevent Ku binding and improve in vitro NER efficiency. Replacement of six contiguous phosphodiester linkages at the 3'-ends of the duplex repair substrate with nuclease-resistant nonionic methylphosphonate linkages resulted in a 280-fold decrease in binding affinity between Ku and the modified duplex. These results are consistent with the published crystal structure of a Ku/DNA complex [Walker et al. (2001) Nature 412, 607-614] and show that the 3'-terminal phosphodiester linkages of linear DNA duplexes are important determinants in DNA end-binding by Ku. Using HeLa whole-cell extracts and a 149-base pair DNA duplex repair substrate, we tested the effects of modification of exposed DNA termini on NER-mediated in vitro excision of a 1,3-GTG-Pt(II) intrastrand cross-link. Methylphosphonate modification at the 3'-ends of the repair substrate resulted in a 1.6-fold increase in excision. Derivatization of the 5'-ends of the duplex with biotin and subsequent conjugation with streptavidin to block Ku binding resulted in a 2.3-fold increase excision. By combining these modifications, we were able to effectively reduce Ku-derived interference of NER excision in vitro and observed a 4.4-fold increase in platinum lesion excision. These modifications are easy to incorporate into synthetic oligonucleotides and may find general utility whenever synthetic linear duplex DNAs are used as substrates to investigate DNA repair in whole-cell extracts.
Mason
Tracey McGregor
TM
Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Johns HopkinsUniversity, 615 North Wolfe Street, Baltimore, Maryland 21205, USA.
Smeaton
Michael B
MB
Cheung
Joyce C Y
JC
Hanakahi
Les A
LA
Miller
Paul S
PS
D000818:Animals; D034961:Antigens, Nuclear / Q000737:chemistry / Q000494:pharmacology; D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D002457:Cell Extracts / Q000737:chemistry; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D009943:Organophosphorus Compounds / Q000737:chemistry; D009944:Organoplatinum Compounds / Q000737:chemistry / Q000494:pharmacology*; D012689:Sequence Homology, Nucleic Acid; D020349:Surface Plasmon Resonance; D013997:Time Factors
D034961:Antigens, Nuclear; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D009944:Organoplatinum Compounds; C032627:methylphosphonic acid; D001710:Biotin; D004247:DNA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen