Biochemistry
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The kinetic parameters (kpol, Kd app) for all possible correct and incorrect pairing between the A, T, G, and C bases were determined for wild-type (WT) rat DNA polymerase beta (pol beta) and the R283A mutant under pre-steady-state kinetic assay conditions. The base substitution fidelities of these two proteins were then determined for all 12 possible mispairs representing the first complete fidelity analysis of polymerases using pre-steady-state kinetics. The results led to several significant findings: (i) For both WT and R283A, the fidelity is determined primarily by kpol (decreases for the incorporation of incorrect nucleotides) and to a small extent by Kd app (increases for the incorporation of incorrect nucleotides). (ii) In general, the fidelity for the Y. ⋯ C (8.6 microM) is distinctly smaller than that of other correct base pairs (41-108 microM). For the R283A mutant, the kpol of G. C is higher by a factor of 15-17.
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Iron-regulatory proteins (IRPs) 1 and 2 are cytosolic RNA-binding proteins that bind to specific stem-loop structures, termed iron-responsive elements (IREs) that are located in the untranslated regions of specific mRNAs encoding proteins involved in iron metabolism. The binding of IRPs to IREs regulates either translation or stabilization of mRNA. Although IRP1 and IRP2 are similar proteins in that they are ubiquitously expressed and are negatively regulated by iron, they are regulated by iron by different mechanisms. ⋯ Like IRP1, the RNA-binding activity of IRP2 was sensitive to inactivation by N-ethylmaleimide (NEM) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), indicating IRP2 contains a cysteine(s) that is (are) necessary for RNA binding. However, unlike IRP1, where reconstitution of the 4Fe-4S cluster resulted in a loss in RNA-binding activity, the RNA-binding activity of IRP2 was unaffected using the same iron treatment. These data suggested that IRP2 does not contain a 4Fe-4S cluster similar to the cluster in IRP1, indicating that they sense iron by different mechanisms.
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The iron responsive element (IRE) is a conserved RNA structure that is found in the 5' UTR of ferritin mRNA and in the 3' UTR of transferrin receptor mRNA. It is the binding site of the iron responsive protein (IRP), and the interaction is part of the regulation of cellular iron metabolism. The IRE six-nucleotide hairpin loop, 5'C1A2G3U4G5N6, is conserved in sequence, and mutations have shown that it is required for IRP binding. ⋯ NMR data suggest that there is hydrogen bonding between C1 and G5 in a tertiary interaction across the loop. A model structure, produced by MC-SYM/energy minimization, illustrates the conformational flexibility of U4 and C6, which appear to exhibit considerable local motion in solution. NMR data indicate that the position of G3 is not well defined, leading to two families of loop structures.
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Integration Host Factor (IHF) is a sequence-specific DNA-bending protein that is proposed to interact with DNA primarily through the minor groove. We have used various chemical probes [(+)-CC-1065, a minor-groove-specific agent that alkylates N3 of adenine and traps bends into the minor groove; pluramycin, a minor-major-groove threading intercalator that alkylates N7 of guanine; KMnO4, which reacts more strongly with bases in denatured DNA] to gain more information on the interaction of IHF with the H' site of phage lambda. In addition to the 13-bp core consensus recognition element present at all IHF binding sites, the H' site also has an upstream AT-rich element that increases the affinity of IHF for this site. ⋯ DNA cyclization studies indicate a large magnitude (approximately 180 degrees) for the IHF-induced bend at the H' site, consistent with > 140 degrees bend estimated by gel electrophoresis methods. These studies suggest that IHF-induced DNA bending is accompanied by the introduction of a DNA node, DNA unwinding, and/or by some other DNA distortion. An enhanced binding and stability of IHF was observed on small circular DNA.
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The T-antigen-induced structural changes of the SV40 replication origin were probed with three DNA-reactive antitumor agents: (+)-CC-1065, bizelesin, and pluramycin. (+)-CC-1065 is an N3 adenine minor groove alkylating agent that selectively reacts with AT-rich DNA sequences with a bent conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a conformation; bizelesin also reacts with the minor groove of AT-rich sequences but is selective for a straight DNA conformation. Pluramycin is an intercalative guanine alkylator whose reactivity is increased by unwinding and decreased by compression of the minor and/or major grooves of DNA. We show that while binding of T-antigen reduced the ability of (+)-CC-1065 to alkylate the AT tract in the SV40 replication origin, it did not interfere with bizelesin modification of the same sequence. ⋯ These data, in combination with the DNase I footprinting results, suggest that T-antigen binding induces a conformational change in the DNA that no longer favors pluramycin intercalation. Based on our results, we propose that T-antigen binds tightly to the upstream region of the AT tract of SV40 replication origin forming double hexamers. In the downstream region, binding of T-antigen to the helically in-phase sites in the GC box region induces DNA bending in the opposite direction of the natural AT tract bending, while simultaneously transforming the naturally bent AT tract DNA into a straight conformation.