Figure 4.
Composition of PARP-5a-pADPr beads and identification of mitotic pADPr-binding proteins. (a) PARP-5a activity is up-regulated during mitosis. GFP-PARP-5a was isolated from lysates of asynchronous (I) and mitosis-arrested (M) HeLa S3 cells using magnetic beads (see Materials and Methods). Beads were washed with high-salt buffer and analyzed by SDS-PAGE and Coomassie blue staining (left) or anti-pADPr Western blot (right). Note much greater pADPr labeling of the M sample. This includes a strong band at the position corresponding to the well of the gel, suggesting that heavily pADPr-labeled proteins failed to enter the gel. The beads shown in the M lane are typical of those used in Figure 3. The numbers to the left show the molecular weights (kDa) of the markers used in all the gels. (b) Identification of pADPr-binding proteins. Beads like those shown in panel a (M) were incubated with mitotic lysate at 4°C and washed with 0.3 M NaCl buffer. These beads were split in two; one-half was analyzed without further treatment (5a + M-lys), the other was treated with a pADPr glycohydrolase (ARH3) and washed (5a + M-lys + ARH3). Note a number of bands that are strongly labeled with Coomassie or anti-pADPr in the lane not treated with glycohydrolase. Most of these were removed by glycohydrolase, indicating they bind to pADPr, and not the beads themselves, or by unmodified PARP-5a. (c) Cell cycle specificity of pADPr-binding proteins. PARP-5a-pADPr beads were made in mitotic lysate and washed with 500 mM NaCl in lysate buffer and then incubated with buffer alone, lysate from asynchronous (I), or mitosis-arrested (M) cells. Note that some of the heaviest bands are similar between the two cell cycle states, but in general the profiles are very different. (d) Drug specificity and requirement for NAD+. pADPr-binding proteins were isolated from cells arrested in mitosis using drugs that inhibit kinesin-5, S-tritl-l-cysteine (STC), or microtubules, nocodazole (NOC). As another test of pADPr requirement, NAD+ was omitted from the both the initial and second extract incubations (left lanes) or added at 500 μM (right lanes). Note that omission of NAD+ caused loss of most of the candidate pADPr-binding proteins. The profile of these proteins was nearly identical for the two drug arrests. (e) Comparison of pADPr and RNA-binding proteins in mitotic lysate. GFP-PARP-5a-pADPr beads were prepared as in panel a. The gel lane labeled 5a shows these beads alone. RNA beads were prepared by incubating biotinylated RNA (Ambion) with streptavidin beads. Both beads were incubated with mitotic lysate, washed, and analyzed by SDS-PAGE. Note that pADPr-coated beads recruited a similar band pattern to b, c (M), and d. RNA beads recruited what appeared to be some similar proteins by band mobility, but that had an overall very different protein profile. (f) Example of band cutting for LC/MS analysis. The boxes indicate bands from the rightmost lane in the gel shown in panel e that were excised for LC/MS analysis. This type of analysis was performed twice, in addition to some LC/MS runs on total pADPr-binding proteins. See Table 1 for the identity of the most abundant protein in each excised band.