Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Incubation of extracts of Cp-1-infected Streptococcus pneumoniae with [alpha-32P]dATP produced a labeled treatment with micrococcal nuclease and sensitive to treatment with proteinase K. Incubation of the 32P-labeled protein with 5 M piperidine for 4 h at 50 degrees C released 5'-dAMP, indicating that a covalent complex between the terminal protein and 5'-dAMP was formed in vitro. When the four deoxynucleoside triphosphates were included in the reaction mixture, a labeled complex of slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels than the terminal protein-dAMP complex was also found, indicating that the Cp-1 terminal protein-dAMP complex can be elongated and, therefore, that it is an initiation complex. Treatment of the 32P-labeled terminal protein-dAMP complex with 5.8 M HCl at 110 degrees C for 2 h yielded phosphothreonine. These results, together with the resistance of the terminal protein-DNA linkage to hydroxylamine, suggest that the Cp-1 terminal protein is covalently linked to the DNA through a phosphoester bond between L-threonine and 5'-dAMP, namely, a O-5'-deoxyadenylyl-L-threonine bond.
 ...
PMID:Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5'-dAMP. 308 36

B. subtilis phage phi 29 has a terminal protein, p3, covalently linked to the 5' ends of the DNA through a phosphodiester bond between a serine residue and 5'-dAMP. This protein acts as a primer in DNA replication by forming an initiation complex with the 5'-terminal nucleotide dAMP. The amino acid sequence of the terminal protein, deduced from the nucleotide sequence of gene 3, showed the presence of 18 serine residues in a total of 266 amino acids. In this paper we have identified the serine involved in the linkage with the DNA as the residue 232, located close to the C-terminus of the molecule. This result was obtained by amino acid analysis of the peptide that remains linked to the DNA after proteinase K digestion of the terminal protein-phi 29 DNA complex and automated Edman degradation of the corresponding [125I]-labeled tryptic peptide. Prediction of the secondary structure of the terminal protein suggested that the serine residue involved in the linkage with the DNA is placed in a beta-turn, probably located on the external part of the molecule, as indicated by hydropathic values.
 ...
PMID:Location of the serine residue involved in the linkage between the terminal protein and the DNA of phage phi 29. 393 46

In the preceeding paper (Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA. This phi X A protein-[32P]DNA complex was the product of the reinitiation reaction. The phi X A protein-[32P]DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures. In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed. The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin. The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence. The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed. Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios. The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized. Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2. In contrast, peptide T2 was unaffected by digestion with proteinase K. These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA. The peptide-mononucleotide complexes T1-[32P]pdA and T2-[32P]pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl. In each case, the major 32P-labeled products were identified as [32P] phosphotyrosine and [32P]Pi. This indicates that each active site of the phi X A protein participates in a phosphodiester linkage between a tyrosyl moiety of the protein and the 5' position of dAMP.
 ...
PMID:Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex. 623 16

Cell-free extracts prepared from phi 29 and M2-infected Bacillus subtilis cells catalyse the formation of complexes between terminal protein and [alpha-32P]-dAMP in the presence of [alpha-32P]-dATP, MgCl2, ATP, and phage DNA with terminal protein covalently linked at both the 5'ends. The complex formation does not take place when proteinase K-treated DNA is added or when uninfected extract is used. The phi 29 complex thus formed is smaller than the M2 complex, primarily due to the different molecular weights of the respective terminal proteins. Extracts prepared from cells infected with suppressor-sensitive mutants of genes 2 or 3 of phi 29 or genes G or E of M2 do not support complex formation. When the pair of extracts of phi 29 or M2-infected cells are mixed, however, formation of the complex takes place as a result of in vitro complementation. These results indicate that the complex formation observed in vitro reflects in vivo initiation of phage DNA replication. The product of gene 2 of phi 29 may be the enzyme that catalyses formation of the complex.
 ...
PMID:In vitro initiation of bacteriophage phi 29 and M2 DNA replication: genes required for formation of a complex between the terminal protein and 5'dAMP. 631 Mar 50

Incubation of extracts of phi 29-infected Bacillus subtilis with [alpha-32P]dATP produced a labeled protein having the electrophoretic mobility of p3, the 5'-terminal protein of phi 29 DNA. The reaction product was resistant to treatment with micrococcal nuclease, phosphatase, and RNases A and T1 and sensitive to proteinase K. Incubation of the 32P-labeled protein with piperidine under conditions in which the phi 29 DNA-protein p3 linkage is hydrolyzed released 5'-dAMP. The reaction with [alpha-32P]dATP was strongly inhibited by anti-p3 serum and required the preence of phi 29 DNA-protein p3 complex; no reaction took place with proteinase K-treated phi29 DNA. These results, together with those of acid hydrolysis and partial proteolysis, indicated that a covalent complex between protein p3 and 5'-dAMP is formed in vitro. The initiation complex (protein p3-dAMP) formed in the presence of 0.5 microM [alpha-32P]dATP can be elongated by addition of 40 microM dNTPs. Treatment with piperidine of the product elongated in the presence of 2',3'-dideoxycytidine 5'-triphosphate released the expected oligonucleotides, 9 and 12 bases long, taking into account the sequence at the left and right DNA ends, respectively.
 ...
PMID:Initiation of phage phi 29 DNA replication in vitro: formation of a covalent complex between the terminal protein, p3, and 5'-dAMP. 681 61