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DNA-Binding Proteins and DNA-Synthesizing Enzymes in Eukaryotes
Published in Lubomir S. Hnilica, Chromosomal Nonhistone Proteins, 2018
DNA joining activities have been measured by a variety of assay methods: (1) the change in sedimentation coefficient after covalently joining the DNA fragments, (2) conversion of internally located 5′-32p-labeled phosphoryl groups to a form resistant to alkaline phosphatase, (3) linkage of one polynulceotide chain to a second one attached to cellulose, and (4) the conversion of 3H-labeled d(A-T) copolymer, chain length of approximately 1000 nucleotides, with 3′-hydroxyl and 5′-phosphoryl termini, to a circular molecule resistant to exonuclease III.
The Use of Molecular Hybridization Techniques as Tools to Evaluate Hepatic Fibrogenesis
Published in Marcos Rojkind, Connective Tissue in Health and Disease, 2017
Mark Α. Zern, Mark J. Czaja, Francis R. Weiner
Most recently, considerable attention has been focused on the delineation of trans-acting factors which may be essential for accurate gene transcription. A series of proteins have been found which bind to specific regions of the eukaryotic promoter and seem to regulate transcription.92,93 Alterations in such proteins have been shown to regulate the expression of several genes that are active in the liver including metallothionein.94 Much of the work which explores the interactions of trans-acting factors and the collagen genes has been done by deCrombrugghe and co-workers. They have used a series of techniques, including the exonuclease III assay, DNase I footprinting, and the DNA gel retardation assay to delineate the relationship of nuclear proteins to the promoter region of the collagen genes.95,96 These techniques can certainly be applied to the regulation of the collagen genes in hepatic fibrosis.
Molecular radiobiology and the origins of the base excision repair pathway: an historical perspective
Published in International Journal of Radiation Biology, 2023
The 5′ AP endonucleases not only cleave the DNA back-bone at AP sites but also contain both phosphatase and diesterase activities that are required to remove blocking groups from the 3′ termini of single strand breaks produced directly by ionizing radiation or by the AP lyases present in the glycosylases described above (Demple and Harrison 1994) (Figure 2). Also, as described in the previous Section, the AP endonucleases can initiate the repair of radiation-induced ‘alkali-labile lesions’ or abasic sites which is how they were originally identified (Demple and Harrison 1994). These AP endonucleases cleave on the 5′ side of the abasic site leaving a 3′ hydroxyl and a 5′ deoxyribose bordering the strand break. The AP activities identified in E. coli are exonuclease III (exo III, Xth) (Richardson and Kornberg 1964; Weiss 1976; Gossard and Verly 1978) and endonuclease IV (endo IV, Nfo) (Ljungquist 1977). Unlike endo IV (Ljungquist 1977), the activity of exo III is stimulated by magnesium (Rogers and Weiss 1980) and also contains 3′-5′ exonuclease (Richardson and Kornberg 1964) and RNase H (Rogers and Weiss 1980) activities. Exo III accounts for about 90% of the AP activity in E. coli but the two activities can substitute for one another thus double mutants xth nfo are hypersensitive to the cytotoxic effects of ionizing radiation (Cunningham et al. 1986; Zhang et al. 1992).
Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma
Published in Journal of Extracellular Vesicles, 2018
Tatyana Vagner, Cristiana Spinelli, Valentina R. Minciacchi, Leonora Balaj, Mandana Zandian, Andrew Conley, Andries Zijlstra, Michael R. Freeman, Francesca Demichelis, Subhajyoti De, Edwin M. Posadas, Hisashi Tanaka, Dolores Di Vizio
We further characterized the DNA enclosed in PC3 cell-derived EVs. First, we found that differences in DNA content of L-EVs and S-EVs were not due to differences in total amount of cargo in L-EVs and S-EVs. Although L-EVs and S-EVs isolated from the same PC3 cells contained nearly equal amounts of protein, L-EVs contained consistently more DNA than S-EVs (Figure 1(e)), confirming that the DNA is included mostly in the L-EV fraction. Additionally, PC3 cell-derived EVs contained both single stranded (ss) and double stranded (ds) DNA at a 5:1 ratio, and the total amount of DNA was markedly higher in L-EVs (Figure 1(f)). To determine if EV DNA was enclosed within EVs or co-precipitated with them during the centrifugation, EVs were treated with DNAse I and Exonuclease III prior to lysis to digest extravesicular DNA. Following this treatment, the total amount of EV DNA was reduced by 35% in L-EVs and by 51% in S-EVs; however, the ssDNA:dsDNA ratio remained the same.
The sequence preference of gamma radiation-induced DNA damage as determined by a polymerase stop assay
Published in International Journal of Radiation Biology, 2019
Megan E. Hardie, Vincent Murray
Gamma radiation can also produce apurinc/apyrimidinic (AP, also known as abasic) sites, oxidized pyrimidine sites, and oxidized purine lesions in human cellular DNA (Sutherland et al. 2002, 2003). These sites can be recognized using a variety of enzymes that cleave next to the lesions. These enzymes include endonuclease IV for AP and oxidized AP sites (Häring et al. 1994; Sutherland et al. 2001; Georgakilas et al. 2004); exonuclease III, endonuclease III (Endlich and Linn 1985; Nakamura and Swenberg 1999) and Ape I for AP sites (Endlich and Linn 1985; Demple et al. 1991). These lesion-specific enzymes have been utilized to detect modified bases (Milligan et al. 2000) and bistranded clusters formed after IR treatment (Prise et al. 1999; Sutherland et al. 2000).