scholarly journals Genetic Mapping of the Amino-Terminal Domain of Bacteriophage T4 DNA Polymerase

Genetics ◽  
1987 ◽  
Vol 115 (3) ◽  
pp. 393-403
Author(s):  
Melanie B Hughes ◽  
Arthur M F Yee ◽  
Myra Dawson ◽  
Jim Karam
Keyword(s):  
Dna Polymerase ◽  
Structural Gene ◽  
Base Pair ◽  
Hot Spots ◽  
Bacteriophage T4 ◽  
Multifunctional Protein ◽  
Amino Terminal ◽  
Preliminary Examination ◽  
Amino Terminal Domain ◽  
Ts Mutations

ABSTRACT The DNA polymerase of bacteriophage T4 is a multifunctional enzyme that harbors DNA-binding, DNA-synthesizing and exonucleolytic activities. We have cloned in bacterial plasmids about 99% of the structural gene for this enzyme (T4 gene 43). The gene was cloned in six contiguous 5'-terminal DNA fragments that defined seven intragenic mapping regions. Escherichia coli hosts harboring recombinant plasmids carrying the gene 43 subsegments were used in marker-rescue experiments that assigned a large number of ts and nonsense polymerase mutations to different physical domains of the structural gene. Conspicuously, only one missense mutation in a large collection of mutants mapped in the 5'-terminal 450 base-pair segment of the approximately 2700 base-pair gene. To test if this indicated a DNA polymerase domain that is relatively noncritical for biological activity, we mutagenized a recombinant plasmid carrying this 5'-terminal region and generated new conditional-lethal mutations that mapped therein. We identified five new ts sites, some having mutated at high frequency (nitrosoguanidine hot spots). New ts mutations were also isolated in phage genes 62 and 44, which map upstream of gene 43 on the T4 chromosome. A preliminary examination of physiological consequences of the ts gene 43 mutations showed that they exhibit effects similar to those of ts lesions that map in other gene 43 segments: some were mutators, some derepressed gene 43 protein synthesis and they varied in the severity of their effects on T4-induced DNA synthesis at nonpermissive temperatures. The availability of the gene 43 clones should make it possible to isolate a variety of lesions that affect different activities of the T4 DNA polymerase and help to define the different domains of this multifunctional protein.

scholarly journals Nuclear localization of the adenovirus DNA-binding protein: requirement for two signals and complementation during viral infection.

1989 ◽  
Vol 9 (10) ◽  
pp. 4372-4380 ◽  
Author(s):  
N Morin ◽  
C Delsert ◽  
D F Klessig
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Viral Infection ◽  
Nuclear Localization ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Multifunctional Protein ◽  
Nuclear Localization Signals ◽  
Amino Terminal ◽  
Amino Terminal Domain

The adenovirus DNA-binding protein (DBP) is an abundant multifunctional protein located primarily in the nuclei of infected cells. To define sequences involved in nuclear transport of DBP, a series of point and small deletion mutants were constructed via oligonucleotide-directed mutagenesis. Two short stretches of basic amino acids located in the amino-terminal domain (amino acids 42 to 46 and 84 to 89) were identified. Their importance, however, depended on the context in which DBP was expressed. Disruption of either site prevented nuclear localization after transient expression in transfected 293 cells, implying that two nuclear localization signals are necessary for transport of this nuclear protein. In contrast, the mutant DBPs synthesized during viral infection were located either primarily in the nucleus or in the nucleus and cytoplasm, depending on the mutation and the stage of the viral infection. Thus, the nuclear localization defect could be complemented by viral infection, perhaps through the interaction of the mutant polypeptide with a virus-encoded or -induced factor(s).

Nuclear localization of the adenovirus DNA-binding protein: requirement for two signals and complementation during viral infection

1989 ◽  
Vol 9 (10) ◽  
pp. 4372-4380
Author(s):  
N Morin ◽  
C Delsert ◽  
D F Klessig
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Viral Infection ◽  
Nuclear Localization ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Multifunctional Protein ◽  
Nuclear Localization Signals ◽  
Amino Terminal ◽  
Amino Terminal Domain

The adenovirus DNA-binding protein (DBP) is an abundant multifunctional protein located primarily in the nuclei of infected cells. To define sequences involved in nuclear transport of DBP, a series of point and small deletion mutants were constructed via oligonucleotide-directed mutagenesis. Two short stretches of basic amino acids located in the amino-terminal domain (amino acids 42 to 46 and 84 to 89) were identified. Their importance, however, depended on the context in which DBP was expressed. Disruption of either site prevented nuclear localization after transient expression in transfected 293 cells, implying that two nuclear localization signals are necessary for transport of this nuclear protein. In contrast, the mutant DBPs synthesized during viral infection were located either primarily in the nucleus or in the nucleus and cytoplasm, depending on the mutation and the stage of the viral infection. Thus, the nuclear localization defect could be complemented by viral infection, perhaps through the interaction of the mutant polypeptide with a virus-encoded or -induced factor(s).

scholarly journals The solution structure of the amino-terminal domain of human DNA polymerase ε subunit B is homologous to C-domains of AAA+ proteins

2008 ◽  
Vol 36 (15) ◽  
pp. 5102-5110 ◽  
Author(s):  
Tarmo Nuutinen ◽  
Helena Tossavainen ◽  
Kai Fredriksson ◽  
Päivi Pirilä ◽  
Perttu Permi ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Solution Structure ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Human Dna ◽  
Amino Terminal Domain ◽  
Subunit B

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

scholarly journals Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase.

Genetics ◽  
1990 ◽  
Vol 124 (2) ◽  
pp. 213-220 ◽  
Author(s):  
L J Reha-Krantz
Keyword(s):  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Rnase H ◽  
Ribonuclease H ◽  
Temperature Sensitive ◽  
Polymerase Gene ◽  
Dna Polymerase I ◽  
E Coli ◽  
Intragenic Complementation ◽  
Polymerase I

Abstract Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.

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