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Regulation of the α2-Macroglobulin Gene
Published in Andrzej Mackiewicz, Irving Kushner, Heinz Baumann, Acute Phase Proteins, 2020
Friedemann Horn, Ursula M. Wegenka, Peter C. Heinrich
Nuclear extracts from Hep G2 cells or rat livers were prepared according to Dignam et al.24 Oligonucleotides or DNA fragments were labeled by filling in 5′ DNA overhangs with the Klenow fragment of DNA polymerase using [α32P]dATR One to 5 μg of protein of nuclear extracts were incubated for 10 min with about 10 fmol (5000 cpm) of the probes in gel-shift incubation buffer [10 mM Hepes, pH 7.8, 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 5 mM dithiothreitol, 0.7 mM phenylmethyl sulfonylfluoride, 1 mg/ml bovine serum albumin, 0.1 to 0.2 mg/ml poly(dIdC)] at room temperature. The DNA-protein complexes formed were separated by electrophoresis on a 4% polyacrylamide gel as described.25 The gels were dried and exposed overnight to an X-ray film.
In situ Hybridization Histochemistry
Published in Edythe D. London, Imaging Drug Action in the Brain, 2017
Martin K.-H. Schofer, James P. Herman, Stanley J. Watson
Nick translation and random primer extension are the most commonly used methods for labeling cDNA probes. Nick translation involves incubation of double-stranded probe molecule (separated from any bacterial sequences by restriction digestion-electrophoresis) with DNase I and Eschericha coli DNA polymerase I. The DNase creates nicks (base excisions) in the cDNA, which is both extended on the 5′ side by the exonuclease activity and filled in on the 3′ side by the polymerase activity of the DNA polymerase I. Radioactive nucleotides included in the translation mix are incorporated during the filling-in process. Random priming, on the other hand, takes advantage of the ability of oligonucleotides to prime DNA synthesis on single-stranded templates. S ingle-stranded cDNAs (again free of bacterial sequences) are incubated with random single-stranded DNA fragments 6 to 12 nucleotides in length in the presence of the Klenow fragment of DNA polymerase I and labeled and unlabeled nucleotides. Generally, random priming introduces a larger proportion of radioactive nucleotides into probes than does nick translation, resulting in two to tenfold higher specific activities (Sambrook et al., 1989).
Polynuclear Platinum Drugs
Published in Astrid Sigel, Helmut Sigel, Metal Ions in Biological Systems, 2004
DNA modified by these trifunctional compounds is able to bind and crosslink BamHI, a sequence-specific DNA-binding protein which recognizes the palindromic sequence GGATCC and also very efficiently binds and crosslinks SP1, a sequence-specific Zn finger protein that induces a bend in the DNA upon binding [73]. Two representative non-sequence-specific DNA-binding proteins, the Klenow fragment from DNA polymerase I, and Klenow exonuclease minus (which has been mutated to remove the 3′-5′ proofreading domain) both bind modified DNA and effectively crosslink to the DNA. Interestingly, preliminary studies also show that HMG domain proteins bind only weakly to DNA modified by the trinuclear complexes. This would be expected since the nature of the interstrand crosslink will be essentially similar to the bifunctional case. The observation of ternary DNA-protein complex formation with SP-1 is therefore noteworthy as it may help to distinguish subtle aspects of protein contact. The SP-1 protein belongs to a family of zinc-finger based transcription factors whose recognition sequences are GC boxes and which bend DNA upon binding [74]. The formation of metal-DNA-protein ternary complexes raises the possibility of “suicide” lesions which may irreversibly sequester a repair protein or transcription factor [75]. The trifunctional agents may find use as protein-targeting drugs and as probes for conformational effects on DNA-protein interactions. DNA-protein crosslinking agents may have a host of possible applications, including identification of contact sites, isolation of weakly bound proteins within a multiprotein complex, as well as representing an attractive target for che-motherapeutic intervention.
Potential therapeutic targets for Mpox: the evidence to date
Published in Expert Opinion on Therapeutic Targets, 2023
Siddappa N Byrareddy, Kalicharan Sharma, Shrikesh Sachdev, Athreya S. Reddy, Arpan Acharya, Kaylee M. Klaustermeier, Christian L Lorson, Kamal Singh
VACV DNA genome replication is conducted by a holoenzyme consisting of multiple proteins [25]. An essential component of this holoenzyme is E9, the DNA-dependent DNA polymerase belonging to B family DNA polymerases. The MPXV genome encodes F8L (OPG71) [2], also a B family DNA-dependent DNA polymerase [27], which shares ~ 98% identity with VACV E9. The first B family DNA polymerase (RB69) structure showed an overall architecture of this class of enzymes [28]. This structure showed a canonical polymerase domain consisting of the Thumb, Palm, and Fingers subdomains, as seen in the structure of the Klenow Fragment (KF) of E. coli DNA polymerase I [29]. A notable difference between these polymerases is the relative position of the 3’ − 5’ exonuclease domain, which is ~ 180° opposite to that in KF relative to the polymerase active site. Subsequent crystal structures of the RB69 polymerase showed that residues of a β-hairpin positioned in the major groove of the template-primer played a role in the partitioning of primer to the 3’ − 5’ exonuclease site upon mismatch nucleotide incorporation [28,30–32]. Indeed, a resistance mutation on topologically similar β-hairpin in poxviruses’ DNA polymerase showed the relevance of the resistance mechanism of nucleotide analogs mediated by 3’ − 5’ exonuclease function (discussed below).
Polymerization-sensitive switch-on monomer for terminal transferase activity assay
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Bhagwan S. Batule, Chang Yeol Lee, Ki Soo Park, Hyun Gyu Park
TdT-primer (5′-AATACAACCTCTCA-3′) used in the study was synthesized by Genotech Co. (Daejeon, South Korea). TdT, phi29 DNA polymerase, exonuclease I (Exo I), Klenow fragment (exo-), Vent (exo-) DNA polymerase, T4 DNA ligase and T4 DNA polymerase were purchased from New England Biolabs Inc. (Beverly, MA), and i-pfu DNA polymerase was purchased from iNtRON Biotechnology (Seongnam, Korea). BODIPY-ATP and FeCl3·6H2O were purchased from Invitrogen (Carlsbad, CA) and Sigma-Aldrich (St. Louis, MO), respectively. All other chemicals were of analytical grade and used without further purification. Ultrapure DNase/RNase-free distilled water purchased from Bioneer® (Daejeon, Korea) was used in all experiments.