Steps in the reaction mechanism of the Haemophilus influenzae Rf restriction endonuclease.
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Biomedical subjects
Publications and source records attributed to A Piekarowicz.
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DNA extracted from phage N3 grown in Haemophilus influenzae strain JC9 was examined by velocity and equilibrium centrifugation, electron microscopy and thermal melting curves. This double stranded DNA, molecular weight 26X10(6) daltons, has continuous single strands at 13X10(6) daltons as shown by alkaline sucrose gradient centrifugation. The duplex form readily aggregates in neutral salt solutions at even moderate ionic strength. Aggregation may be due to N3 DNA having "sticky" ends. CsCl equilibrium centrifugation measurements and thermal melting curves indicate a G+C content of 43 mol %.
Intracellular events following infection of competent Haemophilus influenzae cells by N3 phage or transfection by DNA from phage were examined. After infection by whole phage three forms of intracellular phage DNA were observed by sedimentation velocity analysis. These forms are probably twisted circles, open circles and linear duplexes. In transfection only about 15% of the phage DNA is efficiently taken up by the competent cells. After entry of phage DNA into wild-type cells in transfection the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. These presumably concatamer forms are generated by recombination. In strain rec-1 the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in the wild-type cells. Since rec-1 is transfected with much lower efficiency than the wild-type our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for efficient transfection. These results also show that although phage N3 codes for its own recombination system it cannot operate in the early stages of transfection and succesful transfection is entirely dependent upon the host recombination system.
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Haemophilus influenzae Rf 232, showing the phenomena of restriction and modification, contains an endonuclease that inactivates in vitro the biological activity of DNAs lacking the strain-specific modification. This specific restriction endonuclease has been purified to near homogeneity by a procedure that includes DNA-agarose chromatography. This highly purified enzyme requires ATP and Mg2+ for activity and is stimulated by S-adenosylmethionine. The enzyme seems to cleave DNA at well-defined sites, since it produces a specific pattern of bands upon agarose gel electrophoresis. The enzyme has no ATPase activity. A methylase activity is observed in the course of the endonucleolytic reaction, which probably protects some of the DNA sites from cleavage.
Haemophilus influenzae Rd9 lysogenic for temperate bacteriophage N3 was found to be virtually nontransformable and nontransfectable. This inhibition of transformation and transfection was due partly to the decreased capacity of competent lysogenic cells for irreversible binding of deoxyribonucleic acid (DNA) and partly to some events taking place after adsorption of the DNA. The unadsorbed DNA was not degraded by the competent lysogenic cells.
The restriction endonucleases of type I and II from Haemophilus influenzae were studied for their activity on transforming and transfecting DNA. Type I restriction enzyme from Haemophilus influenzae Rf, which requires adenosine 5'-triphosphate, reduced the size of unmodified bacterial DNA from 66x106 daltons to approximately 18x106 daltons and did not attack modified DNA. The action of this enzyme gives only a low level of inactivation of single and linked markers in the transforming DNA. In contrast the HP1c1 phage DNA was drastically inactivated by this enzyme. The endoR.Hind III degrades the ummodified bacterial DNA but the segments generated by this enzyme are still capable of being integrated in transformation. The enzyme has no activity on HP1c1 phage DNA.
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Transfection of H. influenzae with N3 bacteriophage DNA shows a dependence on concentration of DNA and a sigmoidal shape of uptake of DNA. The efficiency of transfection is decreased in the two types of recombination-defective strains of H. influenzae; rec1- and rec2-. The fact that N3 bacteriophage can code for its own recombination system but transfection is dependent on host recombation system allows the conclusion that the primary recombination during transfection with N3 phage DNA is mediated exclusively by the bacterial recombination system.
A restriction endonuclease has been partially purified from Haemophilus influenzae Rf232 containing the genetically determined system of restriction and modification of DNA. The enzyme requires ATP for the degradation of transfecting phage DNA.
A strain of Haemophilus influenzae, called hpm- inhibits the growth of phage HP1c1 but not S2. This inhibition is overcome by HP1c1ph mutants. Phage HP1c1 adsorbs normally to hpm- cells but only a small fraction of infected cells produce phage with a normal burst size or become lysogenic. When hpm- strains lysogenic for HP1c1 are induced, 100 percent of the cells yield phage. There is no degradation of phage DNA after infection of hpm- cells and HP1c1 can normally grow when its DNA is introduced into hpm- by transfection. The most probable explanation is that in hpm- cells the penetration of phage DNA is blocked. The hpm- property behaves as as unstable mutation.
In Haemophilus influenzae strains only the type 1 of the restriction endonucleases have an in vivo effect on phage and bacterial transforming DNA. The type 2 of restriction endonucleases which act very efficiently in vitro are completely inactive in vivo. The reasons for this inactivity is unknown.
Further investigations of the instability of restriction and modification properties of H. influenzae Rd strain were carried out. It has been shown that the instable properties of hsd Hind1 system are maintained even after transfer of this system to another H. influenzae strain. The expression of hsd Hind1 system is very sensitive to various physiological changes which do not influence the other hsd systems present in the same Rd strain. The instability of hsd Hind1 system is postulated to be connected with some regulator gene(s).
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