PARP-2, a Novel DNA-Damage Dependent Poly ADP-Ribose Polymerase

PARP-2, a Novel DNA-Damage Dependent Poly(ADP-Ribose) Polymerase 

AiFinity purified mouse PARP-2 (mPARP-2) overproduced in the Sf9 insea cells/baculovirus system displays DNA dam e-dependent PARP activity. Using DNAse I treated DNA as a coactivator we estimated a Km of 130 (iM which represents an affinity for NAD 2.6-fold lower than hPARP-1 (50)lM).The kaJKmiatio (323 s M ) is 18 times lower than that of hPARP-1, a value in good reement with the 5-10% residual activity found in PARP-T cell extracts stimulated by DNA strand breaks.  

The DNA-binding domain of mPARP-2 (aa 1-65 Fig. 2) was identified on the basis of its capacity to bind damped DNA in a Southwestern assay. Using precisely defined DNA ends, we demonstrated that purified mPARP-2 binds specifically to a gap of one nucleotide (F. 3A) and protects about 10 nucleotides. In contrast to PARP-1, PARP-2 does not bind to a break (Fig. 3B). The affinity of PARP-2 for the inside of the double helix can also be visualized by electron microscopy (Fig. 3C) where the protein accumulates and seems to displace one of the two DNA strands (arrows) when a break is present. 

In the absence of any protein acceptor, PARP-2 can modify itself in vitro. Most of the radioactive label is found associated with domain E (aa 64-198) that is not only the PARP-2 automodification domain but also the intetaaive interface with partners like PARP-1, XRCCl, DNA pol P, and DNA Ligase III. Domain E is also involved in PARP-2 dimerization.  

From reconstitution experiments where either purified PARP-1 or PARP-2 were mixed with purified nuclei from PARP-1 deficient cells (Fig. 3D), it can be inferred that PARP-1 preferentially poly(ADP-ribosyl)ates the linker histone HI, whereas PARP-2 modifies preferentially the core histone H2B. Therefore, PARP-1 and PARP-2 have different targets both in DNA and in chromatin further indicating that, as chromatin modifiers, they play specific functions. 

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