Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.25.1 (deoxyribonuclease)
 1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphorylation is a major post-translational regulatory mechanism and plays a key role in transduction of mitogenic signals in cell proliferation. The role of phosphorylation and dephosphorylation in regulating the activities of a multiprotein DNA polymerase alpha complex was examined. Treatment of the HeLa cell multiprotein DNA polymerase alpha with calf intestinal alkaline phosphatase resulted in the inactivation of DNA polymerase alpha and DNA primase but had no effect on deoxyribonuclease- and primer-recognition proteins. A protein kinase co-purified with the multiprotein DNA polymerase alpha and was partially purified from HeLa cells. The partially purified kinase was active in phosphorylating dephosphorylated polymerase alpha and used casein and histones as exogenous substrates. This study demonstrates that phosphorylation-dephosphorylation may have modulated the activities of DNA replicative enzymes and suggests a role for specific phosphatases and kinases in this process.
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PMID:Phosphorylation of HeLa cell multiprotein DNA polymerase alpha complex: impact on activity and partial purification of the associated kinase. 256 5

We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.
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PMID:Cloning and assembly of PCR products using modified primers and DNA repair enzymes. 938 51

A common feature of DNA repair enzymes is their ability to recognize the damage independently of sequence in which they are found. The presence of a flipped out base inserted into the protein in several DNA-enzyme complexes suggests a contribution to enzyme specificity. Molecular simulations of damaged DNA indicate that the damage produces changes in DNA structure and changes the dynamics of DNA bending. The reduced bending force constant can be used by the enzyme to induce DNA bending and facilitate base flipping. We show that a thymine dimer (TD) containing DNA requires less energy to bend, lowering the barrier for base flipping. On the other hand, bending in DNA with U-G mismatch is affected only by a small amount and flipping is not enhanced significantly. T4 endonuclease V (endoV), which recognizes TD, utilizes the reduced barrier for flipping as a specific recognition element. In uracil DNA glycosylase (UDG), which recognizes U-G mismatches, base flipping is not enhanced and recognition is encoded in a highly specific binding pocket for the flipped base. Simulations of UDG and endoV in complex with damaged DNA provide insight into the essential elements of the catalytic mechanism. Calculations of pKas of active site residues in endoV and endoV-DNA complex show that the pKa, of the N-terminus is reduced from 8.01 to 6.52 while that of Glu-23 increases from 1.52 to 7.82. Thus, the key catalytic residues are in their neutral form. The simulations also show that Glu-23 is also H-bonded to O4' of the 5'-TD enhancing the nucleophilic attack on Cl and that Arg-26 enhances the hydrolysis by electrostatic stabilization but does not participate in proton transfer. In the enzyme-substrate complex of UDG, the role of electrostatic stabilization is played by His-268, whose pKa increases to 7.1 from 4.9 in the free enzyme. The pKa of Asp-145, the other important catalytic residue, remains around 4.2 in the free enzyme and in the complex. Thus, it can not act as a proton acceptor. In the complex the 3'-phosphate of uracil is stabilized next to Asp-145 by two bridging water molecules. Such a configuration activates one water molecule to act as a proton acceptor to produce a stabilizing hydronium ion and the other as a proton donor to produce the nucleophilic hydroxide. It appears that DNA glycosylases share commonalties in recognition of damage but differ in their catalytic mechanisms.
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PMID:Specificity of damage recognition and catalysis of DNA repair. 1081 3

This work identifies novel structure-function relationships between Tn5 transposase (Tnp) and its DNA recognition sequence. The Tn5 Tnp-DNA co-crystal structure revealed the protein-DNA contacts of the post-cleavage complex (Davies, D. R., Goryshin, I. Y., Reznikoff, W. S., and Rayment, I. (2000) Science 289, 77-85). One of the most striking features of this complex is the rotation of thymine 2 (T2) away from the DNA helix and into a pocket within the Tnp. This interaction appears similar to the "base flipping" phenomenon found in many DNA repair enzymes such as T4 endonuclease V and uracil DNA glycosylase (Roberts, R. J., and Cheng, X. (1998) Annu. Rev. Biochem. 67, 181-198). To study the biochemical significance of this phenomenon, we mutated the Tnp residues proposed to be involved in stabilizing this interaction and removed the T2 nucleotide to examine which steps in the transposition reaction require T2-Tnp interactions. From this work, we have determined that stacking interactions between T2 and Tnp are critical for efficient transposition in vitro. In addition, our results suggested that T2-Tnp interactions facilitate hairpin formation and hairpin resolution primarily through base stacking and that T2 plays a role in the alignment of the transposon DNA for strand transfer.
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PMID:Mutational analysis of the base flipping event found in Tn5 transposition. 1180 7