Proteolysis of a nucleotide excision repair protein by the 26 S proteasome

The 26 S proteasome degrades a broad spectrum of proteins and interacts with several nucleotide excision repair (NER) proteins, including the complex of Rad4 and Rad23 that binds preferentially to UV-damaged DNA. The rate of NER is increased in yeast strains with mutations in genes encoding subunits of the 26 S proteasome, indicating that it could negatively regulate a repair process. The specific function of the 26 S proteasome in DNA repair is unclear. It might degrade DNA repair proteins after repair is completed or act as a molecular chaperone to promote the assembly or disassembly of the repair complex. In this study, we show that Rad4 is ubiquitylated and that Rad23 can control this process. We also find that ubiquitylated Rad4 is degraded by the 26 S proteasome. However, the interaction of Rad23 with Rad4 is not only to control degradation of Rad4, but also to assist in assembling the NER incision complex at UV-induced cyclobutane pyrimidine dimers. We speculate that, following the completion of DNA repair, specific repair proteins might be degraded by the proteasome to regulate repair.     

Complexities of the DNA base excision repair pathway for repair of oxidative DNA damage.

Oxidative damage represents the most significant insult to organisms because of continuous production of the reactive oxygen species (ROS) in vivo. Oxidative damage in DNA, a critical target of ROS, is repaired primarily via the base excision repair (BER) pathway which appears to be the simplest among the three excision repair pathways. However, it is now evident that although BER can be carried with four or five enzymes in vitro, a large number of proteins, including some required for nucleotide excision repair (NER), are needed for in vivo repair of oxidative damage. Furthermore, BER in transcribed vs. nontranscribed DNA regions requires distinct sets of proteins, as in the case of NER. We propose an additional complexity in repair of replicating vs. nonreplicating DNA. Unlike DNA bulky adducts, the oxidized base lesions could be incorporated in the nascent DNA strand, repair of which may share components of the mismatch repair process. Distinct enzyme specificities are thus warranted for repair of lesions in the parental vs. nascent DNA strand. Repair synthesis may be carried out by DNA polymerase beta or replicative polymerases delta and epsilon. Thus, multiple subpathways are needed for repairing oxidative DNA damage, and the pathway decision may require coordination of the successive steps in repair. Such coordination includes transfer of the product of a DNA glycosylase to AP-endonuclease, the next enzyme in the pathway. Interactions among proteins in the pathway may also reflect such coordination, characterization of which should help elucidate these subpathways and their in vivo regulation.     

BRCA1 and p53: compensatory roles in DNA repair

The BRCA1 breast cancer susceptibility gene has been implicated in many cellular processes, yet its specific mechanism of tumor suppression remains unclear. BRCA1 plays a role in several DNA repair pathways including nucleotide excision repair (NER). Loss of the p53 tumor suppressor gene, a key regulator of NER, is an important and necessary event in the pathogenesis of BRCA1-mutated tumors. Here we discuss the role of BRCA1 and NER in breast cancer and the interactions of BRCA1 with p53 in breast tumorigenesis and suggest approaches for risk assessment and chemotherapeutic management of BRCA1-related breast cancer.     

Xeroderma pigmentosum group F protein binds to Eg5 and is required for proper mitosis: implications for XP-F and XFE

The xeroderma pigmentosum group F-cross-complementing rodent repair deficiency group 1 (XPF-ERCC1) complex is a structure-specific endonuclease involved in nucleotide excision repair (NER) and interstrand cross-link (ICL) repair. Patients with XPF mutations may suffer from two forms of xeroderma pigmentosum (XP): XP-F patients show mild photosensitivity and proneness to skin cancer but rarely show any neurological abnormalities, whereas XFE patients display symptoms of severe XP symptoms, growth retardation and accelerated aging. Xpf knockout mice display accelerated aging and die before weaning. These results suggest that the XPF-ERCC1 complex has additional functions besides NER and ICL repair and is essential for development and growth. In this study, we show a partial colocalization of XPF with mitotic spindles and Eg5. XPF knockdown in cells led to an increase in the frequency of abnormal nuclear morphology and mitosis. Similarly, the frequency of abnormal nuclei and mitosis was increased in XP-F and XFE cells. In addition, we showed that Eg5 enhances the action of XPF-ERCC1 nuclease activity. Taken together, these results suggest that the interaction between XPF and Eg5 plays a role in mitosis and DNA repair and offer new insights into the pathogenesis of XP-F and XFE.     

Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease

The RecQ DNA helicases, human BLM and yeast Sgs1, form a complex with topoisomerase III (Top3) and are thought to act during DNA replication to restart forks that have paused due to DNA damage or topological stress. We have shown previously that yeast cells lacking SGS1 or TOP3 require MMS4 and MUS81 for viability. Here we show that Mms4 and Mus81 form a heterodimeric structure-specific endonuclease that cleaves branched DNA. Both subunits are required for optimal expression, substrate binding, and nuclease activity. Mms4 and Mus81 are conserved proteins related to the Rad1-Rad10 (XPF/ERCC1) endonuclease required for nucleotide excision repair (NER). However, the Mms4-Mus81 endonuclease is 25 times more active on branched duplex DNA and replication fork substrates than simple Y-forms, the preferred substrate for the NER complexes. We also present genetic data that indicate a novel role for Mms4-Mus81 in meiotic recombination. Our results suggest that stalled replication forks are substrates for Mms4-Mus81 cleavage-particularly in the absence of Sgs1 or BLM. Repair of this double-strand break (DSB) by homologous recombination may be responsible for the elevated levels of sister chromatid exchange (SCE) found in BLM(-/-) cells.     

Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer.     

Immunohistochemical Analysis of Nucleotide Excision Repair Factors XPA and XPB in Adult Rat Brain

To study the regional and cellular distribution of xeroderma pigmentosum group A and B (XPA and XPB) proteins, two nucleotide excision repair (NER) factors, in the mammalian brain we used immunohistochemistry and triple fluorescent immunostaining combined with confocal microscope scanning in brain slices of adult rat brain, including the cerebral cortex, striatum, substantia nigra compacta, ventral tegmental area, red nucleus, hippocampus, and cerebellum. Both XPA and XPB proteins were mainly expressed in neurons, because the XPA‐ or XPB‐immunopositive cells were only costained with NeuN, a specific neuronal marker, but not with glial fibrillary acidic acid, a specific astrocyte marker, in the striatum. Furthermore, XPA‐ and XPB‐positive staining were observed in the neuronal nuclei. Such subcellular distribution was consistent with the location of the NER in the cells. This study provides the first evidence that NER factors XPA and XPB exist in the nuclei of neurons in the brain, suggesting that the NER may play important roles in the process of DNA repair in adult brain neurons. Anat Rec, 291:775–780, 2008. © 2008 Wiley‐Liss, Inc.     

Effect of p53 tumor suppressor on nucleotide excision repair in human colon carcinoma cells treated with 4-nitroquinoline 1-oxide

In probing the mechanism of nucleotide excision repair (NER) in response to 4-nitroquinoline 1-oxide (4NQO)-induced DNA damage, the effect of p53 tumor suppressor was investigated. The effect of p53 protein on the repair of damaged DNA was examined by comet assay. Expression of p53 and p21(Waf1/Cip1) proteins was measured by the Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry, respectively. Compared to RKO cells having the wild-type p53 gene, increased cytotoxicity by 4NQO was observed in RKOmp53 cells with a mutation in p53 protein. DNA single strand breaks (SSB), indicative of the DNA repair, were considerably increased in 4NQO-treated RKO cells. Also, the expression of p53 and p21 proteins was significantly increased in 4NQO-treated RKO cells. In RKOmp53 cells, no effect of 4NQO on p21 expression was observed. Our findings suggest that 4NQO-induced NER is p53-dependent and involves up-regulation of its downstream regulator, p21(Waf1/Cip1) proteins.     

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry.     

XPC branch‐point sequence mutations disrupt U2 snRNP binding,resulting in abnormal pre‐mRNA splicing in xeroderma pigmentosum patients

Mutations in two branch‐point sequences (BPS) in intron 3 of the XPC DNA repair gene affect pre‐mRNA splicing in association with xeroderma pigmentosum (XP) with many skin cancers (XP101TMA) or no skin cancer (XP72TMA), respectively. To investigate the mechanism of these abnormalities we now report that transfection of minigenes with these mutations revealed abnormal XPC pre‐mRNA splicing that mimicked pre‐mRNA splicing in the patients' cells. DNA oligonucleotide‐directed RNase H digestion demonstrated that mutations in these BPS disrupt U2 snRNP–BPS interaction. XP101TMA cells had no detectable XPC protein but XP72TMA had 29% of normal levels. A small amount of XPC protein was detected at sites of localized ultraviolet (UV)‐damaged DNA in XP72TMA cells which then recruited other nucleotide excision repair (NER) proteins. In contrast, XP101TMA cells had no detectable recruitment of XPC or other NER proteins. Post‐UV survival and photoproduct assays revealed greater reduction in DNA repair in XP101TMA cells than in XP72TMA. Thus mutations in XPC BPS resulted in disruption of U2 snRNP‐BPS interaction leading to abnormal pre‐mRNA splicing and reduced XPC protein. At the cellular level these changes were associated with features of reduced DNA repair including diminished NER protein recruitment, reduced post‐UV survival and impaired photoproduct removal. Hum Mutat 30:1–9, 2009. Published 2009 Wiley‐Liss, Inc.     

Karyomegalic interstitial nephritis and DNA damage-induced polyploidy in Fan1 nuclease-defective knock-in mice

The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1^nd/nd) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1^nd/nd kidneys are polyploid, and fibroblasts from Fan1^nd/nd mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4–Slx1 nuclease also involved in ICL repair.     

Proficient deoxyribonucleic acid repair of methylation damage in hamster ERCC-gene mutants

Three major pathways, nucleotide excision repair (NER), base excision repair (BER) and O⁶-methylguanine–DNA methyltransferase (MGMT), are responsible for the removal of most adducts to DNA and thus for the survival of cells influenced by deoxyribonucleic acid (DNA) adduct-forming chemicals. We have evaluated host cell reactivation and cell survival of wild type Chinese hamster ovary cells and of mutants in the NER-genes ERCC1, ERCC2, and ERCC4 after treatment with the methylating compounds dimethylsulfate and methylnitrosourea. No effect of the three genes could be demonstrated, i.e., survival and host cell reactivation after methylation damage in the mutants and the wild type cells were similar. Gene-specific repair experiments confirmed the proficient removal of methyl lesions. We conclude that the three nucleotide excision repair genes are immaterial to the repair of methylation damage. This suggests that NER does not play a role in the removal of methylation in mammalian cells and that BER and MGMT are responsible for the survival of such cells, when they are challenged with methylation of DNA.