Chromosomal Localization and cDNA Cloning of the Genes (DDB1 and DDB2) for the p127 and p48 Subunits of a Human Damage-Specific DNA Binding Protein

Dualan, Rachel; Brody, Tom; Keeney, Scott; Nichols, Anne F.; Admon, Arie; Linn, Stuart

doi:10.1006/geno.1995.1215

Cited by 144 publications

(113 citation statements)

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“…This restriction fragment (nucleotides ϩ898 to ϩ1690), containing the G 3 A mutation at ϩ818, was ligated into pTB13 (wild type DDB2 cDNA in pBluescript SK (20)), which had been digested with BsmI and ApaI to remove the corresponding wild type cDNA region. The resulting vector was digested with StuI, releasing the XP2RO cDNA sequence Ϫ62 to ϩ1317, which was inserted into pBacPAK8 (CLONTECH) at the StuI site to produce the transfection vector 2RO-p48/pBacPAK8.…”

Section: Methodsmentioning

confidence: 99%

Human Damage-specific DNA-binding Protein p48

Nichols

Itoh

Graham

et al. 2000

Journal of Biological Chemistry

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126

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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...

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Section: Methodsmentioning

confidence: 99%

Human Damage-specific DNA-binding Protein p48

Nichols

Itoh

Graham

et al. 2000

Journal of Biological Chemistry

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126

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“…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].…”

Section: The Special Case Of Cpd Recognitionmentioning

confidence: 99%

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

Clement

Camenisch

Jia

et al. 2010

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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“…A damage-speci®c DNA binding protein (DDB) is a heterodimer of p127 (the product of the DDB1 gene) and p48 (the product of the DDB2 gene) Dualan et al, 1995). DDB preferentially and tightly binds to 6 ± 4 photoproducts and only marginally to cis-syn cyclobutane pyrimidine dimers, for which the binding a nity is almost equal to that of the undamaged thymidines (Reardon et al, 1993;Fujiwara et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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

2001

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A damage-speci®c DNA binding protein (DDB) activity is absent from a subset (DDB 7 ) of cells from individuals initially classi®ed as group E of xeroderma pigmentosum (XP), a hereditary, photosensitive disease with a high incidence of skin malignancies. In these cases, mutations have been identi®ed in the DDB2 gene (DDB2 7 ) that codes for the small subunit, p48, of the DDB heterodimer. In four DDB2 7 strains, neither p48 nor DDB activity were observed before or after UVirradiation, despite an unusually strong up-regulation of DDB2 mRNA levels after UV-irradiation. In a ®fth strain, XP82TO, p48 was detectable and both DDB2 mRNA and p48 levels were more up-regulated after UVirradiation than in normal primary cells. Moreover, DDB activity also became apparent after irradiation. XP82TO showed very mild clinical manifestations compared with the other DDB 7 patients. These results, coupled with our ®ndings that most, if not all DDB + cells classi®ed as XP-E were misclassi®ed, suggests a direct correlation between DDB2 levels and the XP-E phenotype. Oncogene (2001) 20, 7041 ± 7050.

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Chromosomal Localization and cDNA Cloning of the Genes (DDB1 and DDB2) for the p127 and p48 Subunits of a Human Damage-Specific DNA Binding Protein

Cited by 144 publications

References 0 publications

Human Damage-specific DNA-binding Protein p48

Human Damage-specific DNA-binding Protein p48

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

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

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