No abstract

A series of double mutants carrying one of the thermosensitive mutations for DNA synthesis (dnaA, B, C, D, E, F, and G) and the polAl mutation of DeLucia and Cairns, were constructed. Enzyme activities of DNA Polymerases II and III were measured in each mutant. DNA Polymerase II activity was normal in all strains tested. DNA Polymerase III activity is thermosensitive specifically in those strains having thermosensitive mutations at the dnaE locus. From these results we conclude that DNA Polymerases II and III are independent enzymes and that DNA Polymerase III is an enzyme required for DNA replication in Escherichia coli.The isolation by DeLucia and Cairns (1) of an Escherichia coli mutant that lacks DNA Polymerase I activity (polA1) has prompted many investigations into the nature of the DNA synthetic capacity of such strains. The purification and characterization of DNA Polymerase II has been reported by ourselves (2) and others (3, 4). In addition, we have reported the existence of a third DNA polymerase in E. coli (DNA Polymerase III) (2). A physiological function for these enzymes has not been determined.The viability of cells devoid of measurable DNA Polymerase I activity suggests that this enzyme is not an obligatory component of the DNA replication machinery of E. coli. To determine whether polymerases II and III are essential for replication, we examined the DNA polymerases of E. coli mutants that were temperature-sensitive for DNA replication in an attempt to correlate the genetic lesions with altered DNA polymerase activity in vitro. We will present evidence indicating that DNA Polymerase III is the product of an essential gene mapping at the dnaE locus. MATERIALS AND METHODSThe following bacterial strains were usedt:(1) CRT4637: F-thr-leu-his-strr malA mtlh thidnaAT46 (2) CRT2667: F-his-strr malA thi-polAl supdnaBT266 (3) BT1029: H560thyrendol-polAl dnaB (4) PC22: F-his-strr malA xylh arg mtl-thi-polAl sup dnaC2(5) PC79: F-his-strr malA xyl-mtlh thi-polAl supdnaD7 (6) E5111: F-his-strr malA xylh mtlh arg-thi-suppolAl dnaE511(7) E4860: F-his-str' malA xylh mtlh arg-thi-supdnaE486 (8) E4868: F-his-strr malA xyl-mtlh arg thi-suppolAl dnaE486 (9) BT1026: H560thy-endoI-polAl dnaE (10) BT1040: H560 thy endoI polAl dnaE (11) E1011: F-his-strr malA xyl-mtl-arg-thi-suppolAl dnaFI01 (12) JW207: thy-rha-strrpolAl dnaF101 (13) NY73: leu-thy-metE rifr strr polAl dnaG3 (14) CRT2668: F-B1 his-malA strr sup polAl dna+ (15) JG112: W3110 thy-rha-lac-sup-polAl dna+The isolation of the double mutant dnaB polAl, was described (5

Media. Tris(hydroxymethyl )aminomethane (Tris)-minimal medium contains (per liter): 0.5 g of NaCl, 8.0 g of KCI, 12.2 g of Tris, 1.2 g of NH4C1, 0.8 g of sodium pyruvate, 0.2 g of MgC1,, 0.5 g of (NH4)2-SO4 10H2O, 0.01 g of thiamine, 2 g of glucose, 0.44 g 1381 on July 16, 2020 by guest