Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A.

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB- XP-E cell extracts, but microinjection of the protein into DDB- XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.

Differential expression of pyrimidine dimer-binding proteins in normal and UV light-treated vertebrate cells.

The expression of UV damage-specific DNA-binding proteins was examined in various phylogenetically distant species with differing DNA repair phenotypes. Two distinct constitutive DNA-binding activities, one specific for cyclobutane pyrimidine dimers and the other for non-cyclobutane dimer photoproducts, were detected. The expression of these binding activities was found to be variable throughout the animal kingdom: cold-blooded vertebrates show a constitutive cyclobutane dimer-binding activity exclusively, and primates reveal only non-cyclobutane binding activity. In contrast, birds and marsupials appear to express both types of binding activities. The kinetics of expression (rather than the constitutive presence) of these UV damage-specific DNA-binding activities after UV treatment correlate with the cell's capacity for DNA repair. In addition, cyclobutane pyrimidine dimer-binding activities could be detected only in cells with established photoreactivating activity.

Effects of congenital infection of sheep with border disease virus on myelin proteins.

Border disease (BD) of sheep is caused by a virus in the genus Pestivirus that results in decreased myelination throughout the CNS when acquired congenitally. Pregnant ewes were inoculated with BD virus at 50 days of gestation, and myelin proteins were quantified in several regions of the CNS during prenatal and postnatal development of infected lambs for comparison with age-matched controls. Newborn field-infected lambs were also examined. Myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) were measured by densitometric scanning of western blots. Deficiencies in the myelin proteins were detected as early as 116 days of gestation, and the deficiencies of myelin proteins were most pronounced in the cerebellum at all ages examined. PLP and MBP increased from 10-30% of normal in cerebellar white matter at birth to 40-60% of normal at 6 months, suggesting some catch-up in the amount of compact myelin with development. MAG and CNP were between 70 and 80% of control levels in the cerebellum at birth and at 6 months. Similar results were obtained for the corpus callosum and spinal cord of infected lambs, but the deficiencies of myelin proteins were not as great. A common finding in all regions examined was that MBP and PLP were reduced more than MAG and CNP. This is probably explained by a greater deficit of compact myelin, in which MBP and PLP are localized, than of associated oligodendroglial membranes, in which MAG and CNP are concentrated.(ABSTRACT TRUNCATED AT 250 WORDS)

Translocation of a UV-damaged DNA binding protein into a tight association with chromatin after treatment of mammalian cells with UV light.

A UV-damaged DNA binding protein (UV-DDB) is the major source of UV-damaged DNA binding activity in mammalian cell extracts. This activity is defective in at least some xeroderma pigmentosum group E (XP-E) patients; microinjection of the UV-DDB protein into their fibroblasts corrects nucleotide excision repair (NER). In an in vitro reconstituted NER system, small amounts of UV-DDB stimulate repair synthesis a few fold. After exposure to UV, mammalian cells show an early dose-dependent inhibition of the extractable UV-DDB activity; this inhibition may reflect a tight association of the binding protein with UV-damaged genomic DNA. To investigate the dynamics and location of UV-DDB with respect to damaged chromatin in vivo, we utilized nuclear fractionation and specific antibodies and detected translocation of the p127 component of UV-DDB from a loose to a tight association with chromatinized DNA immediately after UV treatment. A similar redistribution was found for other NER proteins, i.e. XPA, RP-A and PCNA, suggesting their tighter association with genomic DNA after UV. These studies revealed a specific protein-protein interaction between UV-DDB/p127 and RP-A that appears to enhance binding of both proteins to UV-damaged DNA in vitro, providing evidence for the involvement of UV-DDB in the damage-recognition step of NER. Moreover, the kinetics of the reappearance of extractable UV-DDB activity after UV treatment of human cells with differing repair capacities positively correlate with the cell's capacity to repair 6-4 pyrimidine dimers (6-4 PD) in the whole genome, a result consistent with an in vivo role for UV-DDB in recognizing this type of UV lesion.

The bacteriophage P1 HumD protein is a functional homolog of the prokaryotic UmuD'-like proteins and facilitates SOS mutagenesis in Escherichia coli.

The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD'. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD' protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD'-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD' functions in vivo as well as examined HumD's physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive DeltaumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD', HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD'-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo.

A 127 kDa component of a UV-damaged DNA-binding complex, which is defective in some xeroderma pigmentosum group E patients, is homologous to a slime mold protein.

A cDNA which encodes a approximately 127 kDa UV-damaged DNA-binding (UV-DDB) protein with high affinity for (6-4)pyrimidine dimers [Abramic', M., Levine, A.S. & Protic', M., J. Biol. Chem. 266: 22493-22500, 1991] has been isolated from a monkey cell cDNA library. The presence of this protein in complexes bound to UV-damaged DNA was confirmed by immunoblotting. The human cognate of the UV-DDB gene was localized to chromosome 11. UV-DDB mRNA was expressed in all human tissues examined, including cells from two patients with xeroderma pigmentosum (group E) that are deficient in UV-DDB activity, which suggests that the binding defect in these cells may reside in a dysfunctional UV-DDB protein. Database searches have revealed significant homology of the UV-DDB protein sequence with partial sequences of yet uncharacterized proteins from Dictyostelium discoideum (44% identity over 529 amino acids) and Oryza sativa (54% identity over 74 residues). According to our results, the UV-DDB polypeptide belongs to a highly conserved, structurally novel family of proteins that may be involved in the early steps of the UV response, e.g., DNA damage recognition.

Biochemical characterization of human DNA polymerase iota provides clues to its biological function.

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase iota (poliota). The role of poliota within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poliota and to speculate as to how these biochemical properties might relate to its in vivo function.

Novel Escherichia coli umuD' mutants: structure-function insights into SOS mutagenesis.

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD', the function of UmuD' has yet to be determined. In an attempt to elucidate the role of UmuD' in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD' plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD' mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD', three were nonsense mutations that resulted in a truncated UmuD' protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD'. All 17 umuD' mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD'.