Binding kinetics and activity of human poly(ADP-ribose) polymerase-1 on oligo-deoxyribonucleotide substrates.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a mammalian enzyme that attaches long branching chains of ADP-ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP-1 plays an important role in DNA strand-break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single-stranded to double-strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single-strandedness were used for measurements of both PARP-1/DNA binding kinetics and PARP-1's enzyme activities. We found that binding correlated with activity, but was unrelated to single-strandedness of the ODN. Instead, PARP-1 binding and activity were highest on ODNs that modeled a DNA double-strand break (DSB). These results provide support for PARP-1 recognizing and binding DSBs in a manner that is independent of single-stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP-1 binding and PARP-1 auto-poly(ADP-ribosyl)ation activities within the same in vitro system.
Jorgensen
Timothy J
TJ
Departments of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA. tjorge01@georgetown.edu
Chen
Kevin
K
Chasovskikh
Sergey
S
Roy
Rabindra
R
Dritschilo
Anatoly
A
Uren
Aykut
A
D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D012984:Software; D020349:Surface Plasmon Resonance
D004277:DNA, Single-Stranded; D008024:Ligands; D009841:Oligonucleotides; D004247:DNA; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D010100:Oxygen