Uracil incorporation: A source of pulse-labeled DNA fragments in the replication of the <i>Escherichia coli</i> chromosome

Abstract

Uracil is incorporated into newly synthesized DNA by mutants of Escherichia coli with reduced levels of dUTPase (dUTP nucleotidohydrolase; EC 3.6.1.23). Excision-repair of the incorporated uracil results in the generation of labeled DNA fragments that appear after brief pulses with [ 3 H]thymidine [Tye, B-K., Nyman, P.-D., Lehman, I. R., Hochhauser, S. &amp; Weiss, B. (1977) Proc. Natl. Acad. Sci. USA 74, 154-157]. Uracil is also incorporated into the newly synthesized DNA of strains of E. coli that contain normal levels of dUTPase. DNA fragments generated by the postreplication excision-repair of uracil may therefore contribute to the pool of nascent DNA (Okazaki) fragments that normally appear in wild-type strains. Discontinuous DNA replication has been examined in the absence of uracil excision by comparing Okazaki fragments in strains that are defective in DNA polymerase I ( polA - ) and polA - strains that are also defective in uracil N -glycosidase, an enzyme required for the excision-repair of uracil in DNA ( polA - ung - ). Little or no difference was detected in the level of Okazaki fragments in the polA - strain as compared with the polA - ung - strain. Thus, the uracil-induced cleavage of DNA cannot be the sole mechanism for the generation of Okazaki fragments. Mutants that are defective both in dUTPase and in uracil N -glycosidase incorporate uracil into their DNA with a high frequency (up to 1 per 100 nucleotides). These uracil residues, once incorporated, persist in the DNA without an adverse affect on the growth of the cells.

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Affiliated Institutions

• University of Minnesota US
• Stanford University US

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