Human Damage-specific DNA-binding Protein p48

Abstract

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...

BRCA1 Transcriptionally Regulates Damaged DNA Binding Protein (DDB2) In the DNA Repair Response Following UV-Irradiation

“…GM01389 cells contain a L350P change in one allele and an Asn-349 deletion in the second allele of DDB2. 48 These results indicate that DDB2 may be required for cell survival in response to UVC and CDDP.…”

Abnormal regulation of DDB2 gene expression in xeroderma pigmentosum group E strains

“…The mutant XP2/ 3RO and XP82TO p48 peptides were similarly expressed and were localized in the nucleus, but these were de®cient in stimulating the nuclear accumulation of p127 (Shiyanov et al, 1999;Nichols et al, 2000;Liu et al, 2000). By immunoprecipitation criteria, the mutant XP2/3RO p48 fails to form a complex with p127, but the mutant XP82TO p48 could bind to it (Shiyanov et al, 1999).…”

“…Therefore, there is an apparent discrepancy of p48 function between the ability to transport p127 to the nucleus and the coimmunoprecipitate p127. After UV-irradiation XP82TO cells have a low level of DDB activity for which both p48 and p127 are necessary in the nucleus (Shiyanov et al, 1999;Nichols et al, 2000). Therefore, mutant XP82TO p48 seems to be able to bind with p127 to at least some degree, but the binding ability may not be su cient to e ciently transport p127 into the nucleus when the cells are unirradiated and p48 levels are low.…”

“…In spite of this complication, it is clear that with four DDB2 7 cell strains (XP2/3RO, GM01389, and XP82TO) uninduced p48 mRNA levels equaled or exceeded those of the normal IMR-90 cells; only in the Ops1 cells might the levels have been lower ( Figure 4a). After UV-irradiation of normal cells, DDB2 mRNA increases, reaching a maximum of 2 ± 3-fold the basal level after 38 h (Figure 4), and then it decreases back to the basal level ( Figure 4b, Nichols et al, 2000). On the other hand, the mRNA levels in each of the DDB2 7 strains XP2/3RO, GM01389 and XP82TO increased nearly 10-fold after irradiation (Figure 4a), and in XP3RO and XP82TO they continued to increase for up to 72 h after UV-irradiation ( Figure 4b).…”

Tumor-prone phenotype of the DDB2-deficient mice

“…XP is a rare autosomal recessive disease characterized by sun-sensitivity and a high incidence of skin malignancies, and these phenotypes are believed to arise from a deficiency in the NER pathway of DNA repair. The DDB2 gene has been linked to XP-E because patients belonging to the XP-E group were shown to harbor mutations in the DDB2 gene (Nichols et al, , 2000Chu and Chang, 1988;Itoh et al, 2000Itoh et al, , 2003. Despite these studies indicating an involvement of DDB2 in DNA repair, it is not clear how DDB2 participates nucleotide excision repair (NER) because the NER assays work efficiently on naked DNA without DDB2 (Reardon et al, 1993;Kazantsev et al, 1996).…”