Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub1⌬ mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.Oxidative DNA damage and the mutations it causes have been implicated in a number of human diseases, including cancer and neurodegenerative diseases, and are a contributing factor to aging (6,12,35,36). Thus, a thorough understanding of genes involved in the prevention and repair of oxidative DNA damage and its mutagenic consequences is important to our understanding of the mechanisms mitigating these diseases and exacerbating normal degenerative processes associated with aging.Oxidative DNA damage results from the interaction of reactive oxygen species (ROS) with DNA. ROS are produced as by-products of normal aerobic metabolism and by exogenous factors, such as ionizing radiation and chemical oxidants (6,12,35,36). The deleterious consequences of ROS to an organism's genetic material are held in check by proteins that prevent or repair oxidative DNA damage (7,12,13,15,21). Unrepaired oxidative lesions result in increased mutagenesis, lethality, and apoptosis (23,27). A balance between DNA repair and damage prevention mechanisms and ROS production is required to maintain a low spontaneous mutation rate. Factors that increase ROS production, reduce ROS detoxification, or factors that affect repair of oxidative DNA lesions result in increased mutagenesis. This is best demonstrated in Escherichia coli; mutations that inactivate the fpg and mutY genes, whose products repair the predominant oxidative lesion 8-oxoguanine (8-oxoG) and its mispaired intermediate 8-oxoG:A, respectively, result in a mutator phenotype that specifically increases GC3 TA transversion mutagenesis (37).In this study, we screened a human cDNA library and describe the isolation and characterization of the human transcription positive cofactor 4 (PC4) gene as a suppressor of oxidative mutagenesis in the E. coli fpg mutY strain. We demonstrate that PC4 and its Saccharomyces cerevisiae ortholog SUB1 are requi...
YSI/1 is a mutant of the common strain (U1) of tobacco mosaic virus (TMV) which induces a severe yellow mosaic in Nicotiana tabacum instead of the light green/dark green mosaic induced by its parental U1 virus. Although there was less coat protein (CP) in whole leaf extracts of YSI/1-infected leaves than in U1-infected leaves, severalfold more CP was found in the chloroplasts, most of which was associated with the thylakoids. Sequencing the CP genes of both viruses showed the presence of nucleotide differences at viral RNA positions 5770 and 6127, both of which result in amino acid replacements; YSI/1 has an Asp-->Val change at amino acid 19 and a Ser-->Phe change at amino acid 138. A common strain TMV engineered to contain the YSI/1 3' end sequences, including the CP, induced the severe yellow mosaic of the YSI/1 mutant. A chimeric virus with the change only at nucleotide 5770 (amino acid 19) in the CP induced a severe yellow mosaic, showing that this replacement is involved in the induction of chlorosis by YSI/1. A second isolate of the same chimera also induced severe yellow mosaic symptoms; sequencing showed that it had gained the change at nucleotide 6127. However, a chimeric virus with the change only at nucleotide 6127 (amino acid 138) in the CP was unable to induce the severe yellow mosaic.
Sphingosine-1-phosphate lyase insufficiency syndrome (SPLIS) is a rare metabolic disorder caused by inactivating mutations in SGPL1, which is required for the final step of sphingolipid metabolism. SPLIS features include steroid-resistant nephrotic syndrome (SRNS) and impairment of neurological, endocrine, and hematopoietic systems. Many affected individuals die within the first two years. No targeted therapy for SPLIS is available. We hypothesized that SGPL1 gene replacement would address the root cause of SPLIS, thereby serving as a universal treatment for the condition. As proof of concept, we evaluated the efficacy of adeno-associated virus 9-mediated transfer of human SGPL1 (AAV-SPL) given to newborn Sgpl1 KO mice that model SPLIS and die in the first weeks of life. Treatment dramatically prolonged survival and prevented nephrosis, neurodevelopmental delay, anemia, and hypercholesterolemia. STAT3 pathway activation and elevated pro-inflammatory and pro-fibrogenic cytokines observed in KO kidneys were attenuated by treatment. Plasma and tissue sphingolipids were reduced in treated compared to untreated KO pups. SGPL1 expression and activity were measurable for at least 40 weeks. In summary, early AAV-SPL treatment prevents nephrosis, lipidosis and neurological impairment in a mouse model of SPLIS. Our results suggest that SGPL1 gene replacement holds promise as a durable and universal targeted treatment for SPLIS.
Macropinocytosis is a clathrin-independent endocytic pathway impli-
The late expression factor 2 gene (lef-2) of baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been identified as one of the factors essential for origin-dependent DNA replication in transient expression assays and has been shown to be involved in late/very late gene expression. To study the function of lef-2 in the life cycle of AcMNPV, lef-2 knockout and repair bacmids were generated by homologous recombination in Escherichia coli. Growth curve analysis showed that lef-2 was essential for virus production. Interestingly, a DNA replication assay indicated that lef-2 is not required for the initiation of viral DNA replication and that, rather, it is required for the amplification of DNA replication. lef-2 is also required for the expression of late and very late genes, as the expression of these genes was abolished by lef-2 deletion. Temporal and spatial distributions of LEF-2 protein in infected cells were also analyzed, and the data showed that LEF-2 protein was localized to the virogenic stroma in the nuclei of the infected cells. Analysis of purified virus particles revealed that LEF-2 is a viral protein component of both budded and occlusion-derived virions, predominantly in the nucleocapsids of the virus particles. This observation suggests that LEF-2 may be required immediately after virus entry into host cells for efficient viral DNA replication.
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