Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Abstract

Cdk-interacting protein 1 (Cipl) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initlafly observed in complexes containing CDK4, cyclin D, and proliferating celi nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymermse (po1) 8, and these three proteins constitute the p01 6 holoenzyme. Cipl is regulated by the tumor suppressor gene p53, a transcription factor that controls the G1 cell cycle arrest checkpoint in response to DNA damage (6). Cipl is transcriptionally induced by DNA damage in a p53-dependent fashion and may mediate cell cycle arrest (7). The G1 checkpoint is thought to arrest the cell cycle to prevent DNA replication of damaged templates while allowing repair.Although kinases are thought to regulate DNA synthesis, the appearance ofCipl and PCNA in a complex suggested the possibility of a more direct link to replication. PCNA, in conjunction with activator 1 (Al; also known as RF-C), acts as a processivity factor for DNA polymerase (pol) 8 (8-10).Recent studies have shown that PCNA (as a trimer) and the f3 subunit (a dimer) of DNA pol III holoenzyme of Escherichia coli both form a tight clamp that tethers their cognate DNA polymerase to the template and translocates along the DNA with the polymerase during replication (11)(12)(13). Thus any agent that can interact with the sliding clamp might impede the movement of the replication complex.Here we demonstrate that Cipl directly binds PCNA to form a complex that inhibits the activity of the PCNAdependent pol 8 holoenzyme, which reduces the replication of simian virus 40 (SV40) DNA and the elongation of primed DNA templates. However, SV40 DNA replication carried out with the pol 8-independent monopolymerase system was unaffected by Cipl. The inhibitory effects of Cipl were independent of the cdk2-cyclin A complex. MATERIALS AND METHODSPreparation of DNAs and Proteins. (dT)1s18 and (dA)4500 were obtained from Life Sciences Inc.; poly(dA) was annealed to oligo(dT) (20:1) as described (14). Singly primed DNA was prepared by hybridizing a 2-fold molar excess of a 34-nt oligonucleotide to circular single-stranded M13 mp7 (7.2 kb) DNA (containing 10-15% linear molecules) at nucleotide positions 6300-6633. The annealed product was labeled by the incorporation of a single dCMP residue (corresponding to position 6299) using the Klenow fragment of DNA pol I. The resulting product (=2000 cpm/fmol), after phenol extraction, was isolated by filtration through a G-50

Twenty years of p53 research: structural and functional aspects of the p53 protein

“…p21WAF1 directly interacts with PCNA and inhibits PCNA-dependent DNA replication (but not DNA repair) in in vitro systems (Flores-Rozas et al, 1994;Li et al, 1994;Waga et al, 1994). p53 protein has also been involved in directly inhibiting DNA replication through its association with the single-stranded DNA-binding protein complex RPA (Dutta et al, 1993) that is required for DNA unwinding during replication.…”

MyD genes in negative growth control

“…It was found that MyD118 and Gadd45 associate with PCNA Hall et al, 1995;Vairapandi et al, 1996), which plays a central role in DNA repair and DNA replication (Mossi and Hubscher 1998;Prosperi, 1997;Jonsson and Hubscher, 1997). MyD118 and Gadd45 were shown also to associate with the cyclin dependent kinase inhibitor p21 (Vairapandi et al, 1996), known to interact with PCNA and to inhibit DNA replication Li et al, 1994;Flores-Rozas et al, 1994). MyD118 and Gadd45 were also observed to enhance DNA excision repair activity in vitro Vairapandi et al, 1996).…”

Ras induces p21Cip1/Waf1 cyclin kinase inhibitor transcriptionally through Sp1-binding sites

“…p21 has several cellular binding partners, including GADD45 damage response protein (Kearsey et al, 1995) and PCNA (Chen et al, 1995). The sequestration of PCNA by p21 from the replication machinery proteins inhibits DNA replication (Flores-Rozas et al, 1994) and interferes with the interaction of PCNA with DNA methyltransferases (Chuang et al, 1997). The loss of p21 leads to defective damage responses and loss of G 1 and mitotic checkpoint control (Brugarolas et al, 1995;Deng et al, 1995).…”