Mutations Specific to the Xeroderma Pigmentosum Group E Ddb− Phenotype

Abstract

The activity of a damage-specific DNA-binding protein (DDB) is absent from a subset, Ddb ؊ , of cell strains from patients with xeroderma pigmentosum group E (XP-E). DDB is a heterodimer of 127-kDa and 48-kDa subunits. We have now identified single-base mutations in the gene of the 48-kDa subunit in cells from the three known Ddb ؊ individuals, but not in XP-E strains that have the activity. An A 3 G transition causes a K244E change in XP82TO and a G 3 A transition causes an R273H change in XP2RO and XP3RO. No mutations were found in the cDNA of the 127-kDa subunit. Overexpression of p48 in insect cells greatly increases DDB activity in the cells, especially if p127 is jointly overexpressed. These results demonstrate that p48 is required for DNA binding activity, but at the same time necessitate further definition of the genetic basis of XP group E.Xeroderma pigmentosum is a rare human hereditary disease, characterized by a high incidence of skin cancer upon exposure to ultraviolet (UV) light and by defective nucleotide excision repair (1). A damage-specific DNA binding (DDB) 1 activity is absent from cell strains from a minority of individuals carrying XP complementation group E (XP-E) (2-4). This activity has been purified from HeLa cells as a complex of a 127-kDa and a 48-kDa polypeptide (5, 6). Studies with duplex oligonucleotides containing unique UV-photoproducts identified a high affinity of DDB for trans-syn-, Dewar-and pyrimidine (6 -4) dimers (7). p127 alone appears to bind specifically to the UV-damaged DNA; however, the heterodimer binds with a different DNase I footprint, indicating that the heterodimer also binds specifically to damaged DNA (7). cDNA clones of DDB1 (p127) and DDB2 (p48) have been isolated from a diploid human fibroblast cDNA library and sequenced (6). While the p127 sequence is not related to any proteins of known function, the p48 sequence has 18% identity and 33% similarity to that of human CSA (Cockayne Syndrome Group A) (8).Evidence that DDB is involved in DNA repair is generally indirect (9 -12). However, microinjection of purified DDB protein into XP-E cells restores repair DNA synthesis to normal levels in XP-E Ddb Ϫ strains, while not affecting that of XP-E Ddb ϩ strains or of cells from other XP groups (13). A feasible explanation for these observations would be that while a mutation(s) in DDB is responsible for defective nucleotide excision repair in XP-E, Ddb Ϫ and Ddb ϩ strains have mutations in different subunits or different domains of a given subunit of DDB. p127 was the most likely candidate for the location of the mutations, since the large subunit alone apparently is capable of DNA binding (7, 12, 14 -16). In the present study, we sequenced cDNAs of both subunits of DDB, but observed mutations to be only in the p48 subunit of Ddb Ϫ XP-E strains. Individual and co-expression of the DDB subunits in insect cells demonstrated that p48 is required to obtain DNA binding activity. EXPERIMENTAL PROCEDURESCell Strains and Culture-Sf9 insect cells (CRL 1711) and IMR-90 norm...

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

“…This factor has been isolated from tissue extracts in view of its characteristic binding to UV-irradiated DNA [55] and, indeed, UV-DDB displays the highest reported affinity for substrates containing 6-4PPs and CPDs [56][57][58]. UV-DDB consists of p127 (DDB1) and p48 (DDB2), with the small subunit being encoded by the XPE gene [59][60][61]. A recent crystallographic analysis demonstrated that the binding of UV-DDB to UV lesions is entirely mediated by the DDB2 subunit, which accommodates the crosslinked pyrimidines into a specialized binding pocket and inserts a three-amino acid hairpin into the DNA minor groove [62].…”

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

“…Recombinant DDB2 has been demonstrated to bind a variety of DNA structures including 6-4 photoproducts, abasic sites, and two base mismatches with remarkably high affinity and CPD lesions and cisplatin adducts with relatively lower affinity (13)(14)(15). Molecular analysis of XP-E patients revealed genetic defects in the DDB2 gene, which give rise to truncations, misfolding, or a modification of the DNA-binding interface of DDB2 (11,12). In the case of the XP82TO patient, a lysine-to-glutamate point mutation at position 244 (K244E) was observed in DDB2, which results in significantly reduced DNA-binding activity and specificity for damage (11,12).…”

“…Finally, unrepaired, CPDs can be bypassed during DNA replication by specialized DNA polymerases, such as DNA polymerase η (pol η) (2). Mutations in the gene encoding pol η give rise to the eighth complementation group of XP, the XP variant phenotype.Molecular defects in DDB2 lead to a slower loss of UVinduced photoproducts and presentation of the skin cancer prone XP complementation group E (XP-E) (11,12). Recombinant DDB2 has been demonstrated to bind a variety of DNA structures including 6-4 photoproducts, abasic sites, and two base mismatches with remarkably high affinity and CPD lesions and cisplatin adducts with relatively lower affinity (13-15).…”

“…Molecular analysis of XP-E patients revealed genetic defects in the DDB2 gene, which give rise to truncations, misfolding, or a modification of the DNA-binding interface of DDB2 (11,12). In the case of the XP82TO patient, a lysine-to-glutamate point mutation at position 244 (K244E) was observed in DDB2, which results in significantly reduced DNA-binding activity and specificity for damage (11,12).In vivo, UV-DDB is constitutively associated with Cullin4A or 4B and RBX1, forming the CRL4 DDB2 E3 ligase complex (16)(17)(18). In this complex, DDB2 is a DNA damage-recognition factor and functions as an adapter protein which targets the E3 ligase activity to sites of UV-induced photoproducts, promoting Significance UV damage in genomic DNA is identified by the human UVdamaged DNA-binding protein (UV-DDB).…”

“…Molecular defects in DDB2 lead to a slower loss of UVinduced photoproducts and presentation of the skin cancer prone XP complementation group E (XP-E) (11,12). Recombinant DDB2 has been demonstrated to bind a variety of DNA structures including 6-4 photoproducts, abasic sites, and two base mismatches with remarkably high affinity and CPD lesions and cisplatin adducts with relatively lower affinity (13)(14)(15).…”