Disease relevance of XPA

• The xeroderma pigmentosum complementation group A (XP-A) protein, XPA, has recently been expressed in Escherichia coli in a soluble and fully functional form [1].
• Carriage of at least one variant allele for XPA G23A was associated with decreased risk of endometrial cancer [odds ratio (OR), 0.70; 95% confidence interval (95% CI), 0.53-0.93] [2].
• In this study, we used synthetic siRNAs targeted to XPA and ERCC1 and compared their effectiveness in sensitising mismatch repair deficient prostate cancer cell lines to cisplatin and mitomycin C [3].
• We also examined the effect of this XPA-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the crude extract and monopolymerase system [4].
• We have made a replication-defective adenovirus, AdCMV-FlagXPA(59-114), that expresses a truncated form of XPA encompassing amino acids 59-114 sufficient for binding to the excision repair cross-complementing protein 1 (ERCC1)/xeroderma pigmentosum complementation group F (XPF) nuclease essential for making an incision 5' of the damage [5].

High impact information on XPA

• Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA [6].
• We conclude that the XPA-deficient mice strongly mimic the phenotype of humans with xeroderma pigmentosum [6].
• The XPA protein functions in a pre-incision step, the recognition of DNA damage [6].
• To permit the functional analysis of the XPA gene in vivo, we have generated XPA-deficient mice by gene targeting in embryonic stem cells [6].
• RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines [7].

Chemical compound and disease context of XPA

• We have studied the role of nucleotide excision repair (NER) in cisplatin DNA damage-induced apoptotic cell death using both normal human fibroblasts and NER-defective xeroderma pigmentosum (XP) XPA and XPG cells [8].
• Human skin fibroblasts, both DNA repair deficient (xeroderma pigmentosum group A: XPA) and DNA repair normal have been co-transformed with a chimeric gene construct containing human CYP1A1 coding sequences controlled by the cadmium (Cd) ion inducible mouse metallothionein-I promoter and pRSV-NEO, a dominant selectable marker for G418 resistance [9].
• Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels [10].
• By contrast, no DSBs are formed after exposure to ultraviolet light in human XPA cells (from a patient with xeroderma pigmentosum complementation group A), which are unable to excise pyrimidine dimers [11].
• This study was performed to elucidate whether xeroderma pigmentosum complementation group A (XPA) carrier has DNA repair abnormality against sun-exposure and ultraviolet (UV)-mimetic chemical carcinogen 4-nitroquinoline 1-oxide (4NQO) [12].

Biological context of XPA

• Formation of a ternary complex by human XPA, ERCC1, and ERCC4(XPF) excision repair proteins [1].
• In contrast, XPA downregulation did not sensitise either cell line to mitomycin C, and only sensitised DU145 cells to cisplatin [3].
• ERCC1 also binds to XPA protein to make a large protein complex at the site of DNA damage [13].
• XPA inhibited SV40 DNA replication in vitro through its interaction with RPA [4].
• To identify proteins that bind to XPA, we screened a HeLa cDNA library using the yeast two-hybrid system [14].

Anatomical context of XPA

• The XPA protein-bound fraction was tested for specific proteins by an in vitro complementation assay with a panel of cell-free extracts from DNA repair-deficient human and rodent cell lines [1].
• Only the 6 testis tumor cell lines showed significantly lower mean levels of XPA (p = 0.001), XPF (p = 0.001) and ERCC1 (p = 0.004) proteins from the other groups [15].
• Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours [16].
• Activation of NER in Cr(VI)-treated human fibroblasts or lung epithelial H460 cells was manifested by XPC-dependent binding of the XPA protein to the nuclear matrix, which was also observed in UV light-treated (but not oxidant-stressed) cells [17].
• Cells cultured from XPA and XPC patients are hypersensitive to UV light, as a result of malfunctioning DNA repair [18].

Associations of XPA with chemical compounds

• When we stratified the patients according to intravesical Bacillus Calmette-Guerin treatment, we found a significant trend for shorter recurrence-free survival time in patients with variant alleles of XPA or ERCC6 polymorphisms who received Bacillus Calmette-Guerin treatment (log rank test, P = 0.078 and 0.022, respectively) [19].
• Sequence analyses and genetic evidence suggest that zinc is associated with a C4-type motif, C105-X2-C108-X17-C126-X2-C129, located in the minimal DNA binding region of XPA (M98-F219) [20].
• Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc associated minimal DNA-binding domain of XPA (ZnXPA-MBD) show directly, for the first time, that the zinc is coordinated to the sulfur atoms of four cysteine residues with an average Zn-S bond length of 2.34+/-0.01 A [20].
• A stretch of consecutive glutamic acid residues in XPA was needed for binding to ERCC1 [21].
• In vivo, the delta E mutant exhibited an intermediate level of complementation of XPA cells and the delta G mutant exhibited little or no complementation [22].

Physical interactions of XPA

• The XPA-bound fraction complemented cell-free extracts of excision repair cross-complementing 1 (ERCC-1), ERCC-4 (XP-F), and XP-A mutants [1].
• XPC physically interacted with XPA, but failed to stabilize the XPA-damaged DNA complex [23].
• We found that RPA interacts with xeroderma pigmentosum group A complementing protein (XPAC), a protein that specifically recognizes UV-damaged DNA [24].
• Here we report the identification of a novel protein designated XAB2 (XPA-binding protein 2) that was identified by virtue of its ability to interact with XPA, a factor central to both nucleotide excision repair subpathways [25].
• A novel cytoplasmic GTPase XAB1 interacts with DNA repair protein XPA [14].

Enzymatic interactions of XPA

• We showed that XPA was a substrate for in vitro phosphorylation by phosphatidylinositol-3-kinase-related kinase family kinases whereas in cells XPA was phosphorylated in an ATR-dependent manner and stimulated by UV irradiation [26].

Regulatory relationships of XPA

• Furthermore, XPAC inhibited DNA polymerase alpha activity in the presence of RPA but not in RPA's absence [24].
• BPD is cytotoxic to Cd induced CYP1A1 expressing XPA cells at > 10-fold lower doses than it is to Cd induced CYP1A1 expressing DNA repair normal cells [9].
• Effect of XPA gene mutations on UV-induced immunostaining of PCNA in fibroblasts from xeroderma pigmentosum group A patients [27].
• In addition, XPC is necessary to promote a stable binding of XPA to UV-irradiated DNA [28].

Other interactions of XPA

• These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome [29].
• The absence of RPA, XPA or XPG activities leads to an intermediate level of strand separation [30].
• We examined the effect of this XPAC-RPA interaction on in vitro simian virus 40 (SV40) DNA replication catalyzed by the monopolymerase system [24].
• These results suggest that XAB1 is a novel cytoplasmic GTPase involved in nuclear localization of XPA [14].
• In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved [31].

Analytical, diagnostic and therapeutic context of XPA

• This analysis showed an increased risk of refractoriness to chemotherapy in the group of patients with XPA variant alleles (RR = 14; P = .02) [32].
• The levels of the mRNA transcripts for ERCC1, XPF, and XPA were measured in a panel of 14 different human cancer cell lines, using real-time PCR [33].
• DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA [34].
• The human XPA and ERCC1 proteins, which are involved in early steps of nucleotide excision repair of DNA, specifically interacted in an in vitro binding assay and a yeast two-hybrid assay [21].
• Immunoblotting revealed that the testis tumour cells had normal amounts of most NER proteins, but low levels of the xeroderma pigmentosum group A protein (XPA) and the ERCC1-XPF endonuclease complex [16].

References