REGULATION OF THE EXPRESSION OR RECRUITMENT OF COMPONENTS OF THE DNA SYNTHESOME BY POLY(ADP-RIBOSE) POLYMERASE

Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replicati on of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the similar to 40 proteins of the MRC, including DNA polymerase alpha (DNA pol al pha), DNA topoisomerase I (topo I), and proliferating-cell nuclear ant igen (PCNA). Although about equal amounts of MRC-complexed and free fo rms of PCNA were detected by immunoblot analysis of HeLa cell extracts , only the complexed form was poly(ADP-ribosyl)ated, suggesting that p oly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC . NAD inhibited the activity of DNA pol delta in the MRC in a dose-dep endent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibit ory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by charact erizing the complex purified from 3T3-L1 cells before and 24 h after i nduction of a round of DNA replication required for differentiation of these cells; at the latter time point, similar to 95% of the cells ar e in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1. cells depleted of PAR P by antisense RNA expression; these cells do not undergo DNA replicat ion nor terminal differentiation. Both PARP protein and activity and e ssentially all of the DNA pol alpha and delta activities exclusively c osedimented with the MRC fractions from S phase control cells, and wer e not detected in the MRC fractions from PARP-antisense or uninduced c ontrol cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP- antisense cells, they were present in the MRC fraction only from induc ed control cells, indicating that PARP may play a role in their assemb ly into an active DNA synthesome. In contrast, expression of DNA pol a lpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these prote ins. Depletion of PARP also prevented induction of the expression of t he transcription factor E2F-1, which positively regulates transcriptio n of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to prol iferate.