The Expression of Poly(ADP-ribose) Polymerase during Differentiation-Linked DNA Replication Reveals That It Is a Component of the Multiprotein DNA Replication Complex

Abstract

3T3-L1 preadipocytes have been shown to exhibit a transient increase in poly(ADP-ribose) polymerase (PARP) protein and activity, as well as an association of PARP with DNA polymerase alpha, within 12-24 h of exposure to inducers of differentiation, whereas 3T3-L1 cells expressing PARP antisense RNA showed no increase in PARP and are unable to complete the round of DNA replication required for differentiation into adipocytes. The role of PARP in differentiation-linked DNA replication has now been further clarified at both the cellular and enzymological levels. Flow cytometric analysis revealed that control 3T3-L1 cells progressed through one round of DNA replication prior to the onset of terminal differentiation, whereas cells expressing PARP antisense RNA were blocked at the G0/G1 phase of the cell cycle. Confocal microscope image analysis of control S phase cells demonstrated that PARP was localized within distinct intranuclear granular foci associated with DNA replication centers. On the basis of these results, purified replicative complexes from other cell types that had been characterized for their ability to catalyze viral DNA replication in vitro were analyzed for the presence of PARP. PARP exclusively copurified through a series of centrifugation and chromatography steps with core proteins of an 18-21S multiprotein replication complex (MRC) from human HeLa cells, as well as with the corresponding mouse MRC from FM3A cells. The MRC were shown to contain DNA polymerases alpha and delta, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as PCNA, RF-C, and RP-A. Finally, immunoblot analysis of MRCs from both cell types with monoclonal antibodies to poly (ADP-ribose) revealed the presence of approximately 15 poly(ADP-ribosyl)ated proteins, some of which were further confirmed to be DNA polymerase alpha, DNA topoisomerase I, and PCNA by immunoprecipitation experiments. These results suggest that PARP may play a regulatory role within the replicative apparatus as a molecular nick sensor controlling the progression of the replication fork or modulates component replicative enzymes or factors in the complex by directly associating with them or by catalyzing their poly(ADP-ribosyl)ation.

Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol α expression during early S phase

“…Consistent with the¯ow cytometric data, in vivo DNA replication was maximal 20 h after release of wild-type and PARP7/7(+PARP) cells into S phase, whereas negligible [ 3 H]thymidine incorporation was apparent in PARP7/7 during the same time period (data not shown). These results are also consistent with our previous data showing that in vivo DNA replication, as assessed by incorporation of bromodeoxyuridine or [ 3 H]thymidine into newly synthesized DNA, was apparent only in 3T3-L1 control cells 24 h after induction of di erentiation-linked DNA replication, but was not detected in PARP-depleted antisense cells (Simbulan-Rosenthal et al, 1996).…”

“…We have shown that PARP-depleted 3T3-L1 cells expressing PARP antisense RNA fail to di erentiate and to undergo DNA replication that normally precedes di erentiation (Simbulan-Rosenthal et al, 1996;Smulson et al, 1995), indicating that PARP appears to be required for di erentiation-linked DNA replication in these cells. PARP is a component of a multiprotein DNA replication complex (MRC or DNA synthesome) that catalyzes replication of viral DNA in vitro and contains pol a, pol d, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as proliferating-cell nuclear antigen (PCNA), RFC, and RPA.…”

“…PARP is a component of a multiprotein DNA replication complex (MRC or DNA synthesome) that catalyzes replication of viral DNA in vitro and contains pol a, pol d, DNA primase, DNA helicase, DNA ligase, and topoisomerases I and II, as well as accessory proteins such as proliferating-cell nuclear antigen (PCNA), RFC, and RPA. PARP poly(ADP-ribosyl)ates 15 of the *40 MRC component proteins, including pol a, topoisomerase I, and PCNA (Simbulan-Rosenthal et al, 1996).…”

Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro

“…In the absence of NAD, PARP inhibits DNA repair through binding to damaged DNA (26). Other functions proposed for PARP include roles in cellular NAD depletion (27), antirecombination and genomic stability (28), and DNA replication (29). PARP also serves as a marker for the onset of apoptosis, after which it is cleaved by proteases into DNA-binding and catalytic fragments (30).…”

Nucleotide excision repair “a legacy of creativity”

“…as discrete and separate processes. Recently, however, several were found to be members of replication complexes (uracil-N-glycosylase, 5-MeC transferase, PARP, XRCC1) [69][70][71][72]. A newer view could regard familiar processes such as NER as temporary associations made from a larger pool of DNA-interacting proteins.…”