PARP-3 Is a Mono-ADP-ribosylase That Activates PARP-1 in the Absence of DNA

Olga Loseva,Ann-Sofie Jemth,Helen E Bryant,Herwig Schüler,Lari Lehtiö,Tobias Karlberg,Thomas Helleday

doi:10.1074/jbc.m109.077834

Abstract

The PARP-3 protein is closely related to the PARP-1 and PARP-2 proteins, which are involved in DNA repair and genome maintenance. Here, we characterized the biochemical properties of human PARP-3. PARP-3 is able to ADP-ribosylate itself as well as histone H1, a previously unknown substrate for PARP-3. PARP-3 is not activated upon binding to DNA and is a mono-ADP-ribosylase, in contrast to PARP-1 and PARP-2. PARP-3 interacts with PARP-1 and activates PARP-1 in the absence of DNA, resulting in synthesis of polymers of ADP-ribose. The N-terminal WGR domain of PARP-3 is involved in this activation. The functional interaction between PARP-3 and PARP-1 suggests that it may have a role in DNA repair. However, here we report that PARP-3 small interfering RNA-depleted cells are not sensitive to the topoisomerase I poison camptothecin, inducing DNA single-strand breaks, and repair these lesions as efficiently as wild-type cells. Altogether, these results suggest that the interaction between PARP-1 and PARP-3 is unrelated to DNA single-strand break repair.

Highlights

(poly(ADP-ribose) polymerase 1) is an abundant nuclear protein that is activated by DNA strand breaks to modify acceptor proteins with poly(ADP-ribose) (PAR)4 [3]
It was shown that PARP-3 interacts with PARP-1 and co-immunoprecipitates with the catalytic subunit of DNA-dependent protein kinase, DNA ligases III and IV, Ku80, and Ku70, proteins involved in DNA repair [16, 17]
Auto- and Trans-ADP-ribosylation Activity of PARP-3—We investigated the ADP-ribosylation activity of both truncated PARP-3 and full-length PARP-3 using Bio-NADϩ and histone H1 as a substrate

Summary

EXPERIMENTAL PROCEDURES

ADP-ribosylation Assay—Expression and purification of recombinant full-length PARP-3 and truncated PARP-3 (Lys178–His532; tPARP-3) were as described previously [12]. PARP-1 was preincubated with the inhibitors for 5 min, and reactions were started by the addition of Bio-NADϩ and activated DNA. After 2.5, 5, and 10 min of incubation at room temperature, the reactions were stopped by dilution in LDS sample buffer. To check the dependence of PARP-1 activity on the amount of PARP-3 present, 20 nM PARP-1 was incubated with different concentrations of PARP-3 in PARP reaction buffer in the presence of 25 ␮M Bio-NADϩ and 75 ␮M NADϩ and in the absence of DNA for 5 min. Inhibition with meta-Iodobenzylguanidine (MIBG)—250 nM purified PARP-3 was incubated with 12.5 ␮M Bio-NADϩ in PARP reaction buffer and with different concentrations of MIBG for 30 min at room temperature. Hydrolysis with HgCl2—125 nM purified PARP-3 was incubated with 12.5 ␮M Bio-NADϩ in PARP reaction buffer for 30 min at room temperature. Depletion was confirmed by reverse transcription-PCR as described previously [9]

RESULTS

DISCUSSION

Karlberg and Thomas Helleday

ADDITIONS AND CORRECTIONS