Terminal Cross-linking of DNA Catalyzed by an Enzyme System Containing DNA Ligase, DNA Polymerase, and Exonuclease of Bacteriophage T7

1974 ◽  
Vol 52 (6) ◽  
pp. 525-535 ◽  
Author(s):  
Paul Sadowski ◽  
Allison McGeer ◽  
Andrew Becker
Keyword(s):  
Dna Polymerase ◽  
Enzyme System ◽  
Maximal Activity ◽  
Cross Linking ◽  
Dna Ligase ◽  
Escherichia Coli Cells ◽  
Phage Dna ◽  
Phage T7 ◽  
Cross Links ◽  
T7 Exonuclease

An enzymatic activity that terminally cross-links T7 DNA has been detected in extracts from phage T7-infected Escherichia coli cells. This activity required the functional products of gene 1.3 (T7 DNA ligase), gene 5 (T7 DNA polymerase), and gene 6 (T7 exonuclease) as well as Mg2+ ions, the four deoxytriphosphates, and ATP. The cross-linking system could be reconstituted with purified ligase, polymerase, and exonuclease, and maximal activity required the presence of all three enzymes. The system was active on phage DNA which possessed unique termini but not on DNA with non-unique ends.

scholarly journals The human checkpoint sensor and alternative DNA clamp Rad9–Rad1–Hus1 modulates the activity of DNA ligase I, a component of the long-patch base excision repair machinery

2005 ◽  
Vol 389 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Ekaterina SMIRNOVA ◽  
Magali TOUEILLE ◽  
Enni MARKKANEN ◽  
Ulrich HÜBSCHER
Keyword(s):  
Quality Control ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Ligase ◽  
Dna Polymerase Β ◽  
Flap Endonuclease 1 ◽  
Important Target ◽  
Dna Ligase I ◽  
Base Excision

The human checkpoint sensor and alternative clamp Rad9–Rad1–Hus1 can interact with and specifically stimulate DNA ligase I. The very recently described interactions of Rad9–Rad1–Hus1 with MutY DNA glycosylase, DNA polymerase β and Flap endonuclease 1 now complete our view that the long-patch base excision machinery is an important target of the Rad9–Rad1–Hus1 complex, thus enhancing the quality control of DNA.

scholarly journals Chemistry of collagen cross-links: glucose-mediated covalent cross-linking of type-IV collagen in lens capsules

1993 ◽  
Vol 296 (2) ◽  
pp. 489-496 ◽  
Author(s):  
A J Bailey ◽  
T J Sims ◽  
N C Avery ◽  
C A Miles
Keyword(s):  
Type Iv Collagen ◽  
Cross Linking ◽  
Mechanical And Thermal Properties ◽  
Maximum Strain ◽  
Scanning Calorimetry ◽  
Melting Temperatures ◽  
Type Iv ◽  
Collagen Molecules ◽  
Cross Links

The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or ‘strength’) was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.

scholarly journals Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents

1988 ◽  
Vol 255 (3) ◽  
pp. 869-876 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Small Subunit ◽  
Cross Linking ◽  
Minor Effect ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Lysine Residues ◽  
Cross Links ◽  
A Minor ◽  
Chemical Cross Linking

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2′-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.

scholarly journals Deep-sea vent phage DNA polymerase specifically initiates DNA synthesis in the absence of primers

2017 ◽  
Vol 114 (12) ◽  
pp. E2310-E2318 ◽  
Author(s):  
Bin Zhu ◽  
Longfei Wang ◽  
Hitoshi Mitsunobu ◽  
Xueling Lu ◽  
Alfredo J. Hernandez ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deep Sea ◽  
Phage Dna

scholarly journals Native cross-links in collagen fibrils induce resistance to human synovial collagenase

1979 ◽  
Vol 181 (3) ◽  
pp. 639-645 ◽  
Author(s):  
C A Vater ◽  
E D Harris ◽  
R C Siegel
Keyword(s):  
Lysyl Oxidase ◽  
Collagen Fibrils ◽  
Bone Collagen ◽  
Collagen Molecule ◽  
Cross Linking ◽  
Pathological Conditions ◽  
Insoluble Material ◽  
Induce Resistance ◽  
Cross Links

A model system consisting of highly purified lysyl oxidase and reconstituted lathyritic chick bone collagen fibrils was used to study the effect of collagen cross-linking on collagen degradation by mammalian collagenase. The results indicate that synthesis of approx. 0.1 Schiff-base cross-link per collagen molecule results in a 2–3-fold resistance to human synovial collagenase when compared with un-cross-linked controls or samples incubated in the presence of beta-aminopropionitrile to inhibit cross-linking. These results confirm previous studies utilizing artificially cross-linked collagens, or collagens isolated as insoluble material after cross-linking in vivo, and suggest that increased resistance to collagenase may be one of the earliest effects of cross-linking in vivo. The extent of intermolecular cross-linking among collagen fibrils may provide a mechanism for regulating the rate of collagen catabolism relative to synthesis in normal and pathological conditions.

EXPERIMENTAL VALIDATION OF THE CHARLESBY AND HORIKX MODELS APPLIED TO DE-VULCANIZATION OF SULFUR AND PEROXIDE VULCANIZATES OF NR AND EPDM

2016 ◽  
Vol 89 (4) ◽  
pp. 671-688 ◽  
Author(s):  
M. A. L. Verbruggen ◽  
L. van der Does ◽  
W. K. Dierkes ◽  
J. W. M. Noordermeer
Keyword(s):  
Experimental Validation ◽  
Main Chain ◽  
Sol Gel ◽  
Chain Scission ◽  
Cross Linking ◽  
Cross Link ◽  
Practical Reality ◽  
Cross Links ◽  
The Cross ◽  
Theoretical Considerations

ABSTRACT The theoretical model developed by Charlesby to quantify the balance between cross-links creation of polymers and chain scission during radiation cross-linking and further modifications by Horikx to describe network breakdown from aging were merged to characterize the balance of both types of scission on the development of the sol content during de-vulcanization of rubber networks. There are, however, disturbing factors in these theoretical considerations vis-à-vis practical reality. Sulfur- and peroxide-cured NR and EPDM vulcanizates were de-vulcanized under conditions of selective cross-link and random main-chain scissions. Cross-link scission was obtained using thiol-amine reagents for selective cleavage of sulfur cross-links. Random main-chain scission was achieved by heating peroxide vulcanizates of NR with diphenyldisulfide, a method commonly employed for NR reclaiming. An important factor in the analyses of these experiments is the cross-linking index. Its value must be calculated using the sol fraction of the cross-linked network before de-vulcanization to obtain reliable results. The values for the cross-linking index calculated with sol-gel data before de-vulcanization appear to fit the experimentally determined modes of network scission during de-vulcanization very well. This study confirms that the treatment of de-vulcanization data with the merged Charlesby and Horikx models can be used satisfactorily to characterize the de-vulcanization of NR and EPDM vulcanizates.

scholarly journals Localization of the Transglutaminase Cross-Linking Sites in the Bacillus subtilis Spore Coat Protein GerQ

2006 ◽  
Vol 188 (21) ◽  
pp. 7609-7616 ◽  
Author(s):  
Alicia Monroe ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Coat Protein ◽  
Cross Linking ◽  
Spore Coat ◽  
Bacillus Subtilis Spore ◽  
Binding Partners ◽  
Lysine Residues ◽  
Cross Links ◽  
Hydrolysis Of ◽  
Spore Coat Protein

ABSTRACT The Bacillus subtilis spore coat protein GerQ is necessary for the proper localization of CwlJ, an enzyme important in the hydrolysis of the peptidoglycan cortex during spore germination. GerQ is cross-linked into high-molecular-mass complexes in the spore coat late in sporulation, and this cross-linking is largely due to a transglutaminase. This enzyme forms an ε-(γ-glutamyl) lysine isopeptide bond between a lysine donor from one protein and a glutamine acceptor from another protein. In the current work, we have identified the residues in GerQ that are essential for transglutaminase-mediated cross-linking. We show that GerQ is a lysine donor and that any one of three lysine residues near the amino terminus of the protein (K2, K4, or K5) is necessary to form cross-links with binding partners in the spore coat. This leads to the conclusion that all Tgl-dependent GerQ cross-linking takes place via these three lysine residues. However, while the presence of any of these three lysine residues is essential for GerQ cross-linking, they are not essential for the function of GerQ in CwlJ localization.

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