Anne F. Nichols

Mutations Specific to the Xeroderma Pigmentosum Group E Ddb− Phenotype

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 → G transition causes a K244E change in XP82TO and a G → 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.

The Naturally Occurring Mutants of DDB Are Impaired in Stimulating Nuclear Import of the p125 Subunit and E2F1-Activated Transcription

ABSTRACT 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.

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

A damage-specific DNA binding protein (DDB) activity is absent from a subset (DDB−) of cells from individuals initially classified as group E of xeroderma pigmentosum (XP), a hereditary, photosensitive disease with a high incidence of skin malignancies. In these cases, mutations have been identified in the DDB2 gene (DDB2−) that codes for the small subunit, p48, of the DDB heterodimer. In four DDB2− strains, neither p48 nor DDB activity were observed before or after UV-irradiation, despite an unusually strong up-regulation of DDB2 mRNA levels after UV-irradiation. In a fifth strain, XP82TO, p48 was detectable and both DDB2 mRNA and p48 levels were more up-regulated after UV-irradiation than in normal primary cells. Moreover, DDB activity also became apparent after irradiation. XP82TO showed very mild clinical manifestations compared with the other DDB− patients. These results, coupled with our findings that most, if not all DDB+ cells classified as XP-E were misclassified, suggests a direct correlation between DDB2 levels and the XP-E phenotype.

Limitations of the in vitro repair synthesis assay for probing the role of DNA repair in platinum resistance

Several studies have implicated enhanced DNA repair in acquired platinum resistance. To better understand the mechanism of increased repair we have employed an in vitro assay using cell-free extracts from platinum sensitive and resistant murine and human cell lines. Since the platinum resistant murine cell lines used in our previous studies had shown increased repair of diaminocyclohexane(dach)-Pt-DNA adducts while one of the resistant human cell lines did not, we have measured in vitro repair synthesis on DNA damaged by (d,l)-trans-1,2-diaminocyclohexanedichloroplatinum(II) (PtCl2(dach)). The results of this assay were strongly dependent on the method used to calculate repair synthesis activity and appeared to disagree with previous estimates of repair activity in these cell lines. By one method of calculation the in vitro repair synthesis assay underestimated the ratio of repair activities in the resistant versus the sensitive murine cell lines, while by the other method the in vitro assay overestimated the ratio of repair activities in the resistant versus the sensitive human cell lines.