Poly(ADP-ribose) Quantification at the Femtomole Level in Mammalian Cells

Poly(ADP-ribose) binding to Chk1 at stalled replication forks is required for S-phase checkpoint activation

“…The iPOND assay was performed essentially as described 47 Synthesis and fractionation of PAR. PAR was synthesized, purified and HPLC fractionated as described 52,67,68 . Briefly, a reaction mixture containing 150 nM recombinant PARP1 was incubated in 100 mM Tris-HCl pH 7.8, 10 mM MgCl 2 , 10 mM DTT, 60 mg ml À 1 histone H1, 60 mg ml À 1 histone type IIa, 1 mM NAD þ and 50 mg ml À 1 double-stranded activator oligonucleotide 5 0 -GGAATTCC-3 0 for 15 min at 37°C.…”

Poly(ADP-ribose) Reactivates Stalled DNA Topoisomerase I and Induces DNA Strand Break Resealing

“…Automodified or mock-modified enzymes were used freshly for DNA binding/cleavage assays or stored at Ϫ80°C in 50 mM Tris-HCl buffer, pH 7.4, containing 14 mM ␤-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, 20% glycerol. Free poly(ADPribose) was prepared as described previously (15) and their size distribution determined (16).…”

Poly(ADP-ribose) Binds to Specific Domains of p53 and Alters Its DNA Binding Functions

“…Free polymers were isolated by affinity chro-matography on dihydroxyboronate resin. They had an average chain length of 16 -20 residues, with ϳ6% of total ADP-ribose moieties being incorporated in branched polymeric chains (16).…”