The human UV-damaged-DNA binding protein DDB has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E), because a subset of XP-E patients lack the damaged-DNA binding function of DDB. Moreover, the microinjection of purified DDB complements the repair deficiency in XP-E cells lacking DDB. Two naturally occurring XP-E mutations of DDB, 82TO and 2RO, have been characterized. They have single amino acid substitutions (K244E and R273H) within the WD motif of the p48 subunit of DDB, and the mutated proteins lack the damaged-DNA binding activity. In this report, we describe a new function of the p48 subunit of DDB, which reveals additional defects in the function of the XP-E mutants. We show that when the subunits of DDB were expressed individually, p48 localized in the nucleus and p125 localized in the cytoplasm. The coexpression of p125 with p48 resulted in an increased accumulation of p125 in the nucleus, indicating that p48 plays a critical role in the nuclear localization of p125. The mutant forms of p48, 2RO and 82TO, are deficient in stimulating the nuclear accumulation of the p125 subunit of DDB. In addition, the mutant 2RO fails to form a stable complex with the p125 subunit of DDB. Our previous studies indicated that DDB can associate with the transcription factor E2F1 and can function as a transcriptional partner of E2F1. Here we show that the two mutants, while they associate with E2F1 as efficiently as wild-type p48, are severely impaired in stimulating E2F1-activated transcription. This is consistent with our observation that both subunits of DDB are required to stimulate E2F1-activated transcription. The results provide insights into the functions of the subunits of DDB and suggest a possible link between the role of DDB in E2F1-activated transcription and the repair deficiency disease XP-E.The human UV-damaged-DNA binding protein has been linked to the repair deficiency disease xeroderma pigmentosum group E (XP-E). Cells from about 30% of XP-E patients (6 of 19) were shown to lack the damaged-DNA binding activity of DDB (3,27). DDB was originally identified as an activity that binds to UV-damaged DNA (3). It has high affinities for the 6-4 photoproduct (14,29,36). In addition, DDB also binds to cisplatin-modified DNA (14). It has been proposed that the damaged-DNA binding activity of DDB is related to a potential DNA repair function (7,15,18,20,36). The microinjection of purified DDB overcomes the repair deficiencies in cells from XP-E patients lacking the damaged-DNA binding activity of DDB (20, 27). However, purified DDB has no significant effect in nucleotide excision repair assays in vitro (18). A recent study proposed that DDB functions as a repair protein in the context of chromatin structure (27). A repair function of DDB would be consistent with the fact that the p48 subunit of DDB possesses extensive sequence homology with the Cockayne syndrome protein CS-A, which is involved in transcription-coupled DNA repair (2,9,12,19).DDB also possesses a transcriptional functi...

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...

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Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunoblotting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases delta and epsilon. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically.

Damage-specific DNA binding (DDB) activity purifies from HeLa cells as a heterodimer (p127 and p48) and is absent from cells of a subset (Ddb ؊ ) of xeroderma pigmentosum Group E (XPE) patients. Each subunit was overexpressed in insect cells and purified. Both must be present for the damaged DNA band shift characteristic of the HeLa heterodimer. However, overexpressed p48 peptides containing the mutations found in three Ddb ؊ XPE strains are inactive, and wild type p48 restores DDB activity to extracts from a fourth XPE Ddb ؊ strain, GM01389, in which compound heterozygous mutations in DDB2 (p48) lead to a L350P change from one allele and a Asn-349 deletion from the other. Although these results indicate that these mutations are each responsible for the loss of DDB activity, they do not affect nuclear localization of p48. In normal fibroblasts, a 4-fold increase in p48 mRNA amount was observed 38 h after UV irradiation, preceding a similar elevation in p48 protein and DDB activity at 48 h, implying that p48 limits DDB activity in vivo. Because DNA repair is virtually complete before 48 h, a role for DDB other than DNA repair is suggested.The rare human hereditary disease, xeroderma pigmentosum (XP), 1 is characterized biochemically by defective nucleotide excision repair (NER), which manifests clinically as sensitivity to ultraviolet light and a high incidence of skin cancer. Based on fusion studies of cells from XP patients, seven NERdefective complementation groups (A through G) and a postreplication repair-deficient variant group (XPV) have been identified (1, 2). Cell strains from a subset (Ddb Ϫ ) of individuals carrying XP complementation group E (XPE) lack a damage-specific DNA binding (DDB) activity (3-5). Because DDB was reported to recognize many types of DNA lesions (6 -11) and is inducible by treatment with DNA-damaging agents (7, 12, 13), DDB was originally expected to play a role in damage recognition prior to nucleotide excision repair. However, recent NER reconstitution studies have reported that DDB is not required in vitro (14 -16). Nonetheless, microinjection of purified HeLa DDB heterodimer (p127, p48) into XPE cells restores in vivo DNA repair synthesis to normal levels in XPE Ddb Ϫ strains but not in XPE Ddb ϩ strains or in cells from other XP groups (17). Sequencing of the cDNAs that encode the DDB heterodimer have identified single base mutations only in DDB2 (p48) of XPE Ddb Ϫ cells. In the Ddb Ϫ strains, XP2RO and XP3RO, a G 3 A transition at nucleotide ϩ818 causes an R273H change in p48, whereas an A 3 G transition at nucleotide ϩ730 causes a K244E change in XP82TO. Overexpression of wild type p48, but not of p127, in insect cells greatly increases DDB activity in extracts prepared from these cells, indicating that p48 is required for damage-specific DNA binding (18).In the present study, we have reconstituted human DDB activity in an electrophoretic mobility shift assay by combining insect extracts containing individually overexpressed wild type p127 and p48. Both extracts were requir...