Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

Abstract

The p21 Cdn1 protein (cip1/waf1/sdi1) plays an important role as an inhibitor of mammalian cell proliferation in response to DNA damage. By interacting with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues, even though this pathway is PCNA-dependent. We investigated features of p21-resistant NER using human cell extracts. A direct endlabelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, ®lling of the *30 nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap ®lling was uncoupled from incision. Binding p21 to PCNA could also inhibit ®lling of model 30 nt gaps by both puri®ed DNA polymerases d and e. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved with time. This incubation gives p21 the opportunity to associate with other targets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap ®lling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an e cient inhibitor of NER synthesis in cell extracts.

Long-patch base excision repair of apurinic/apyrimidinic site DNA is decreased in mouse embryonic fibroblast cell lines treated with plumbagin: involvement of cyclin-dependent kinase inhibitor p21Waf-1/Cip-1

“…The LP-BER activity was determined with the repair of R-cccDNA (300 ng) by measuring the incorporation of [a-32 P]-dCTP. Photographs of the autoradiogram is representative of two independent experiments p21-mediated decrease in long-patch BER in plumbagin-treated cells AS Jaiswal et al Shivji et al, 1998). The inhibition of DNA synthesis and cell cycle arrest at G 1 /S phase transition or in S phase has been suggested due to inhibition of PCNA function and Cdk/cyclin kinase activity, respectively, by p21 (Rousseau et al, 1999).…”

“…Our target molecules were p21 and PCNA, since both are involved in cell cycle arrest (Cayrol et al, 1998;Dulic et al, 1999;Li et al, 1994b) and DNA repair (reviewed by Dotto, 2000;Jonsson and Hubscher, 1997;Kelman, 1997;Warbrick, 2000;Wilson, 1998). The role of p21 in NER is reported to be through interaction with PCNA; however, it is still unclear whether p21 inhibits (Li et al, 1994a;Shivji et al, 1994Shivji et al, , 1998 or creates resistance to NER (Cooper et al, 1999;Pan et al, 1995). In a recent study, it is suggested that p21 is required for NER at a step located downstream of the recruitment of PCNA to DNA repair sites (Stivala et al, 2001).…”

Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells

“…Calf thymus DNA polymerase ␦ (Pol ␦; a gift from U. Hübscher, University of Zürich) was purified as described (15), and human proliferating cell nuclear antigen (PCNA) was produced in Escherichia coli (16,17). After annealing to the template, the primer was extended with T7 DNA polymerase (Sequenase version 2.0, United States Biochemical) or Pol ␦ with PCNA, as described (18). Reactions were terminated with sequencing stop buffer (98% deionized formamide͞25 mM Tris-borate-EDTA͞0.025% bromophenol blue͞0.025% xylene cyanol) and loaded onto a 14% denaturing polyacrylamide gel for electrophoresis.…”

p21CDKN1A Does Not Interfere with Loading of PCNA at DNA Replication Sites, but Inhibits Subsequent Binding of DNA Polymerase D at the G1/S Phase Transition

“…This effect is obtained not only through CDK inhibition, but also by direct binding to the proliferating cell nuclear antigen (PCNA), thereby interfering with PCNA-dependent DNA synthesis, 3,4 while permitting PCNA-dependent DNA repair. 5,6 The dual effect of p21 on cell cycle regulatory proteins is mediated via distinct interaction sites for cyclinCdk complexes and for PCNA. [7][8][9][10][11] In fact, p21 has a CDK binding site in its N-terminal region (aa 53-58), which is distinct from two cyclin binding sites located in the N-and C-terminal regions, [12][13][14][15] respectively.…”