Multiple pathways of copy control of gamma replicon of R6K: mechanisms both dependent on and independent of cooperativity of interaction of tau protein with DNA affect the copy number.
The ability of a replication initiator protein to promote intermolecular pairing of two replication origins resulting in the turning off of the origin pair has been called handcuffing. We have endeavored to test the validity of the handcuffing model by isolating two mutant forms of the tau initiator protein of R6K that elicit high copy number phenotype. We have discovered that one mutant called tau 113 yielded a 3.6-fold increase in copy number of a gamma replicon with a concomitant impairment of its ability to loop DNA and to pair binding sites (iterons) in comparison with normal tau, thus supporting the handcuffing model. A second mutant called tau 108, on the other hand, elicited a 3-fold increase in copy number without showing any measurable loss in its ability to loop and pair gamma iterons. Both mutant forms of the wild-type tau protein showed no detectable differences in their affinity of binding to the gamma iterons. Thus, the phenotype of tau 108 is consistent with the proposition that copy number control involves macromolecular interactions other than cooperativity at a distance of tau or interaction of tau with the primary binding sites at gamma. Taken together, the results are consistent with the notion that tau-mediated handcuffing is a mechanism, but not the only mechanism, of copy control in R6K. Interaction of tau with host proteins is likely to provide additional facets of the copy control mechanism.
Department of Microbiology, Duke University Medical Center, Durham, NC 27710.
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Miron
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Patel
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Bastia
D000595:Amino Acid Sequence; D001483:Base Sequence; D016384:Consensus Sequence; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D007700:Kinetics; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012093:Replicon; D015183:Restriction Mapping; D016875:tau Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D016875:tau Proteins; D003850:Deoxyribonuclease I