Poly(ADP-ribose) polymerase null mouse cells synthesize ADP-ribose polymers.
Poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.30), the only enzyme known to synthesize ADP-ribose polymers from NAD+, is activated in response to DNA strand breaks and functions in the maintenance of genomic integrity. Mice homozygous for a disrupted gene encoding PARP are viable but have severe sensitivity to gamma-radiation and alkylating agents. We demonstrate here that both 3T3 and primary embryo cells derived from PARP-/- mice synthesized ADP-ribose polymers following treatment with the DNA-damaging agent, N-methyl-N'-nitro-N-nitrosoguanidine, despite the fact that no PARP protein was detected in these cells. ADP-ribose polymers isolated from PARP-/- cells were indistinguishable from that of PARP+/+ cells by several criteria. First, they bound to a boronate resin selective for ADP-ribose polymers. Second, treatment of polymers with snake venom phosphodiesterase and alkaline phosphatase yielded ribosyladenosine, a nucleoside diagnostic for the unique ribosyl-ribosyl linkages of ADP-ribose polymers. Third, they were digested by treatment with recombinant poly(ADP-ribose) glycohydrolase, an enzyme highly specific for ADP-ribose polymers. Collectively, these data demonstrate that ADP-ribose polymers are formed in PARP-/- cells in a DNA damage-dependent manner. Because the PARP gene has been disrupted, these results suggest the presence of a previously unreported activity capable of synthesizing ADP-ribose polymers in PARP-/- cells.
Shieh
W M
WM
Department of Clinical Sciences, University of Kentucky, Lexington, Kentucky 40506-0286, USA.
Amé
J C
JC
Wilson
M V
MV
Wang
Z Q
ZQ
Koh
D W
DW
Jacobson
M K
MK
Jacobson
E L
EL
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005838:Genotype; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009153:Mutagens / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis*; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000502:physiology*
D009153:Mutagens; D009243:NAD; D008769:Methylnitronitrosoguanidine; D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases