Role of p21WAF1 in the cellular response to UV

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.     

Role of p21WAF1 in the Cellular Response to UV

UV or gamma irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin-dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21 function.     

The oncogenic phosphatase WIP1 negatively regulates nucleotide excision repair

Nucleotide excision repair (NER) is the only mechanism in humans to repair UV-induced DNA lesions such as pyrimidine (6-4) pyrimidone photoproducts and cyclobutane pyrimidine dimers (CPDs). In response to UV damage, the ataxia telangiectasia mutated and Rad3-related (ATR) kinase phosphorylates and activates several downstream effector proteins, such as p53 and XPA, to arrest cell cycle progression, stimulate DNA repair, or initiate apoptosis. However, following the completion of DNA repair, there must be active mechanisms that restore the cell to a prestressed homeostatic state. An important part of this recovery must include a process to reduce p53 and NER activity as well as to remove repair protein complexes from the DNA damage sites. Since activation of the damage response occurs in part through phosphorylation, phosphatases are obvious candidates as homeostatic regulators of the DNA damage and repair responses. Therefore, we investigated whether the serine/threonine wild-type p53-induced phosphatase 1 (WIP1/PPM1D) might regulate NER. WIP1 overexpression inhibits the kinetics of NER and CPD repair, whereas WIP1 depletion enhances NER kinetics and CPD repair. This NER suppression is dependent on WIP1 phosphatase activity, as phosphatase-dead WIP1 mutants failed to inhibit NER. Moreover, WIP1 suppresses the kinetics of UV-induced damage repair largely through effects on NER, as XPD-deficient cells are not further suppressed in repairing UV damage by overexpressed WIP1. Wip1 null mice quickly repair their CPD and undergo less UV-induced apoptosis than their wild-type counterparts. In vitro phosphatase assays identify XPA and XPC as two potential WIP1 targets in the NER pathway. Thus WIP1 may suppress NER kinetics by dephosphorylating and inactivating XPA and XPC and other NER proteins and regulators after UV-induced DNA damage is repaired.     

Efficient repair of bulky anti-BPDE DNA adducts from non-transcribed DNA strand requires functional p53 but not p21(waf1/cip1) and pRb

Wild-type p53 protein is known to regulate the global genomic repair (GGR), removing bulky chemical DNA adducts as well as cyclobutane pyrimidine dimers from the genome overall and from non-transcribed strands (NTS) in DNA. To investigate the role of cellular factor(s) relevant to p53 regulated DNA repair processes, we examined the repair kinetics of chemical carcinogen, anti-benzo[a]pyrene-diol epoxide (anti-BPDE), induced bulky DNA adducts in normal human mammary epithelial cells (HMECs) and HMEC transformed by human papillomavirus (HPV)-16E6 or -16E7 oncoproteins, which, respectively targets p53 or pRb proteins for degradation. The results show that the removal of anti-BPDE DNA adducts from the genome overall and NTS by GGR was significantly reduced in HPV-16E6 protein expressing cells as compared to that in normal and HPV-16E7 protein expressing cells, indicating the role of p53 and not pRb in nucleotide excision repair (NER). We further determined the potential effects of the p53-regulated p21(waf1/cip1) gene product in NER in human colon carcinoma, HCT116 cells expressing wild-type p53 but different p21(waf1/cip1) genotypes (p21+/+, p21+/-, p21-/-). The results donot show a discernible difference in the removal of anti-BPDE DNA adducts from the genome overall and the transcribed strand (TS) and NTS irrespective of the presence or absence of p21(waf1/cip1) expression. Based on these results, we suggest that: (i) the wild-type p53 function but not p21(waf1/cip1) expression is necessary for GGR of chemical induced bulky DNA adducts; (ii) the Rb gene product does not play a significant role in NER; and (iii) the modulation of NER by p53 may be independent of its function in the regulation of cell cycle arrest upon chemically induced DNA damage.     

Roles of Rad23 protein in yeast nucleotide excision repair

Nucleotide excision repair (NER) removes many different types of DNA lesions. Most NER proteins are indispensable for repair. In contrast, the yeast Rad23 represents a class of accessory NER proteins, without which NER activity is reduced but not eliminated. In mammals, the complex of HR23B (Rad23 homolog) and XPC (yeast Rad4 homolog) has been suggested to function in the damage recognition step of NER. However, the precise function of Rad23 or HR23B in NER remains unknown. Recently, it was suggested that the primary function of RAD23 protein in NER is its stabilization of XPC protein. Here, we tested the significance of Rad23-mediated Rad4 stabilization in NER, and analyzed the repair and biochemical activities of purified yeast Rad23 protein. Cellular Rad4 was indeed stabilized by Rad23 in the absence of DNA damage. Persistent overexpression of Rad4 in rad23 mutant cells, however, largely failed to complement the ultraviolet sensitivity of the mutant. Consistently, deficient NER in rad23 mutant cell extracts could not be complemented by purified Rad4 protein in vitro. In contrast, partial complementation was observed with purified Rad23 protein. Specific complementation to the level of wild-type repair was achieved by adding purified Rad23 together with small amounts of Rad4 protein to rad23 mutant cell extracts. Purified Rad23 protein was unable to bind to DNA, but stimulated the binding activity of purified Rad4 protein to N-acetyl-2-aminofluorene-damaged DNA. These results support two roles of Rad23 protein in NER: (i) its direct participation in the repair biochemistry, possibly due to its stimulatory activity on Rad4-mediated damage binding/recognition; and (ii) its stabilization of cellular Rad4 protein.     

CP-31398 restores DNA-binding activity to mutant p53 in vitro but does not affect p53 homologs p63 and p73

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effective in vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elements in vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (B(max)) and its affinity (K(d)) for DNA. The compound, however, does not affect the affinity (K(d) value) of wild type p53 for DNA and only increases B(max) slightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.     

p300/CBP acetyl transferases interact with and acetylate the nucleotide excision repair factor XPG

Nucleotide excision repair (NER) is an important DNA repair mechanism through which cells remove bulky DNA lesions. Following DNA damage, the histone acetyltransferase (HAT) p300 (also referred to as lysine acetyltransferase or KAT) is known to associate with proliferating cell nuclear antigen (PCNA), a master regulator of DNA replication and repair processes. This interaction, which results in HAT inhibition, may be dissociated by the cell cycle inhibitor p21^CDKN1A, thereby restoring p300 activity; however, the role of this protein interplay is still unclear. Here, we report that silencing p300 or its homolog CREB-binding protein (CBP) by RNA interference (RNAi) significantly reduces DNA repair synthesis in human fibroblasts. In addition, we determined whether p300 and CBP may associate with and acetylate specific NER factors such as XPG, the 3′-endonuclease that is involved in the incision/excision step and is known to interact with PCNA. Our results show that p300 and CBP interact with XPG, which has been found to be acetylated in vivo. XPG is acetylated by p300 in vitro, and this reaction is inhibited by PCNA. Knocking down both p300/CBP by RNAi or by chemical inhibition with curcumin greatly reduced XPG acetylation, and a concomitant accumulation of the protein at DNA damage sites was observed. The ability of p21 to bind PCNA was found to regulate the interaction between p300 and XPG, and an abnormal accumulation of XPG at DNA damage sites was also found in p21^−/− fibroblasts. These results indicate an additional function of p300/CBP in NER through the acetylation of XPG protein in a PCNA–p21 dependent manner.     

Centrin 2 stimulates nucleotide excision repair by interacting with xeroderma pigmentosum group C protein

Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome nucleotide excision repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed alpha-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.     

DNA repair is activated in early stages of p53-induced apoptosis

p53 is a complex molecule involved in apoptosis, cell cycle arrest, and DNA repair. Since apoptosis may play an important role in deletion of neoplastic cells, an understanding of the mechanism of p53-induced apoptosis may be critical for possible future therapeutic interventions. Recent evidence suggests that p53-induced apoptosis may involve members of the nucleotide excision repair (NER) family, linking these two cellular events. Our work using a temperature-sensitive p53 construct further analyzes p53-induced apoptosis in cultured murine mammary epithelial cells and also suggests that DNA repair plays a role in that process. Although p21 is induced in our system, apoptosis occurs without a detectable preceding G1 cell cycle arrest and independent of cellular alterations brought on by the temperature shift. In addition, clonogenic assays suggest that early stages of p53-induced apoptosis may be reversible upon removal of the apoptosis stimulus. As a possible explanation for this reversibility, our results show that general DNA repair activity increases early in p53-induced apoptosis. We also show that caspase-3 is activated at a timepoint when colony formation begins to drop, suggesting a possible mechanism for the point of no return in p53-induced apoptosis.