Manzoor A. Wani

Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage

Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.

Cellular ubiquitination and proteasomal functions positively modulate mammalian nucleotide excision repair

The ubiquitin‐proteasome pathway is fundamental to synchronized continuation of many cellular processes, for example, cell‐cycle progression, stress response, and cell differentiation. Recent studies have shown that the ubiquitin‐proteasome pathway functions in the regulation of nucleotide excision repair (NER) in yeast. In order to investigate the role of the ubiquitin‐proteasome pathway in the NER of mammalian cells, global genomic repair (GGR), and transcription‐coupled repair (TCR) were examined in a mouse ts20 cell line that harbors a temperature‐sensitive ubiquitin‐activating enzyme (E1). We found that E1 inactivation‐induced ubiquitination deficiency decreased both GGR and TCR, indicating that the ubiquitination system is involved in the optimization of entire NER machinery in mammalian cells. We specifically inhibited the function of 19S proteasome subunit by overexpressing 19S regulatory complex hSug1 or its mutant protein hSug1mk in repair competent human fibroblast, OSU‐2, cells and compared their capacity for NER. The results showed that 19S regulatory complex positively modulates NER in cells. In addition, we treated OSU‐2 cells with the inhibitors of 20S subunit function, MG132 and lactacystin, and demonstrated that the catalytic activity of 20S subunit is also required for efficient NER. Moreover, the UV‐induced recruitment of repair factor xeroderma pigmentosum protein C (XPC) to damage sites was negatively affected by treatment of repair competent cells with MG132. Taken together, we conclude that the ubiquitin‐proteasome pathway has a positive regulatory role for optimal NER capacity in mammalian cells and appears to act through facilitating the recruitment of repair factors to DNA damage sites.

p53-degradation by HPV-16 E6 preferentially affects the removal of cyclobutane pyrimidine dimers from non-transcribed strand and sensitizes mammary epithelial cells to UV-irradiation.

Nucleotide excision repair (NER), the most versatile and ubiquitous mechanism for DNA repair, operates to remove many types of DNA base lesions. We have studied the role of p53 function in modulating the repair of DNA damage following UV irradiation in normal and p53-compromised human mammary epithelial cells (HMEC). The effect of UV-induced DNA damage on cellular cytotoxicity and apoptosis was determined in conjunction with global, gene- and strand-specific repair. Cytotoxicity studies, using clonogenic survival and MTT assays, showed that HPV-16 E6-expressing HMEC were more UV sensitive than p53-WT cell lines. High apoptotic index obtained with p53-compromised cells was in conformity to both the low clonogenic survival and the low cellular viability. No discernible differences in the formation of initial UV-induced cyclobutane pyrimidine dimers (CPD) were observed in the cell lines of varying p53 functional status. However, the extent and the rate of damage removal from genome overall were highest for p53-WT cells. Further examination of strand-specific repair in the p53 gene revealed that the removal of CPD in the non-transcribed strand (NTS) was slower in p53-compromised cells compared to the normal p53-WT cell lines. These results suggest that loss of p53 function, in the absence of other genetic alterations, decreased both overall amount of CPD repaired and their removal rate from the genome. Additionally, normal function of p53 is required for the repair of the NTS, but not of the transcribed strand (TS) in genomic DNA in human epithelial cells. Thus, failure of quantitative removal of CPD by global genomic repair (GGR), due to loss of p53 function, causes the enhanced UV sensitivity and increased damage-induced apoptosis via a p53-independent pathway. Nevertheless, recovery of cells from UV damage requires normal p53 function and efficient GGR.

Efficient repair of bulky anti-BPDE DNA adducts from non-transcribed DNA strand requires functional p53 but not p21waf1/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.

β2-Microglobulin Deficient Mice Catabolize IgG More Rapidly Than FcRn-α-Chain Deficient Mice

FcRn, a nonclassical MHC-I protein bound to β2-microglobulin (β2m), diverts IgG and albumin from an intracellular degradative fate, prolonging the half-lives of both. While knockout mouse strains lacking either FcRn-α-chain (AK) or β2m (BK) show much shorter half-lives of IgG and albumin than normal mice, the plasma IgG half-life in the BK and AK strains is different, being shorter in the BK strain. Since β2m does not affect the IgG production rate, we tested whether an additional β2m-associated mechanism protects IgG from catabolism. First, we compared the fractional disappearance rate in plasma of an intravenous dose of radioiodinated IgG in a mouse strain deficient in both FcRn-α-chain and β2m (ABK), in the two parental knockout strains (AK and BK), and in the background wild-type (WT) strain. We found that IgG survived longer in the β2m-expressing AK strain than in the β2m-lacking ABK and BK strains, whereas the IgG half-lives between the ABK and BK strains were identical. Then we compared endogenous concentrations of four typical plasma proteins among the four strains and found that steady-state plasma concentrations of both IgG and albumin were higher in the AK strain than in either the BK or the ABK strain. These results suggest that a β2m-associated effect other than FcRn prolongs the survival of both IgG and albumin, although leaky gene transcription in the AK strain cannot be ruled out.

Modulation of transcriptional activity of p53 by ultraviolet radiation: linkage between p53 pathway and DNA repair through damage recognition.

The increase in the p53 activity in response to DNA damage is thought to be one of the important mechanisms by which p53 contributes to transcriptional activation of p21wafl, mdm2, and other downstream regulatory genes. To investigate the p53 response to ultraviolet (UV) type of DNA damage, p53 protein level, its transcriptional activity and in vivo ubiquitination were compared in repair‐proficient normal human fibroblasts (NHFs) and repair‐deficient xeroderma pigmentosum (XP) group A and group C (XP‐C) fibroblasts subsequent to irradiation with UV light. Accumulation of p53 protein level was observed with increasing UV doses in all the cell lines; however, discordance between p53 and p21waf1 and mdm2 levels was observed in NHF and XP‐A cells. Induction of p21waf1 and mdm2 was inhibited by UV irradiation, requiring higher doses in NHF and lower doses in XP‐A cells. However, inhibition of p21waf1 and mdm2 induction was not observed in XP‐C cells. Ubiquitin‐p53 conjugates could be detected in irradiated or unirradiated NHF and XP‐A cells but not in XP‐C cells irradiated with 30 and 50 J/m2 UV light. Using a p53 reporter assay, p53 transcriptional activities were found to be induced by 10 J/m2 UV exposure and dramatically inhibited with increasing UV doses in NHF cells. Compared with repair‐proficient NHF cells, UV inhibition of p53 transcriptional activity was relatively more sensitive in XP‐A cells but resistant in XP‐C cells. These results indicate that DNA damage by UV, in addition to inducing p53, acts as a trigger for inhibition of p53 transcriptional activity. Overall, recognition of DNA damage links both p53 induction and p53 degradation to DNA repair mechanisms. Mol. Carcinog. 28:215–224, 2000.