Transient accumulation of Okazaki fragments as a result of uracil incorporation into nascent DNA.

Abstract

Strains of Escherichia coli with a mutation in the sof(dnaS) locus show a higher than normal frequency of recombination (are hyper rec) and incorporate label into short (4-5S) DNA (Fig. 1). Second, an excision-repair system detects and removes uracil residues that may have escaped the action of dUTPase and were misincorporated into DNA. Lindahl (4) has described an N-glycosidase that catalyzes the cleavage of the uracildeoxyribose linkage in DNA, and nucleases, acting at the apyrinidinic acid site, might excise that region of the backbone (5, 6); the gap could be filled in by DNA polymerase I and DNA ligase to complete the repair process (7). Gates and Linn § have very recently identified an endonuclease that may also serve in removal of uracil residues by its specific capacity to hydrolyze uracil-containing DNA.A defect in dUTPase would be expected to produce an increase in the intracellular pool of dUTP, and in addition, to block the predominant pathway of thymidine nucleotide biosynthesis, both of which should lead to an increased level of uracil in DNA. However, a group of dUTPase mutants recently isolated by Hochhauser and Weiss (8)

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

“…Since extracts of ung dug strains lack detectable uracil-initiated BER [16], it would appear that Ung and Dug are the sole activities in E. coli capable of removing uracil from DNA. Thus, use of CY11 allows determination of the amount of uracil that escaped cleavage by dUTP pyrophosphatase and was incorporated into nascent DNA under various growth conditions [3]. We consistently observed that the level of uracil in DNA extracted from saturated cultures of CY11 was lower than that from early log cultures (Fig.…”

“…Although the DNA polymerases of E. coli do not effectively discriminate dUTP from dTTP, little dUTP finds its way into E. coli DNA because of the action of dUTP pyrophosphatase, which hydrolyzes dUTP to dUMP and PP i [10]. In early studies of dut mutants, it was anticipated that the dut mutant DNA would contain uracil [10]; however, the nascent DNA of these mutants was found to be exceptionally fragmented [3]. Such fragmentation was consistent with the excision of transiently incorporated uracil by uracil-DNA glycosylase (Ung) and subsequent incision of the Ung-generated abasic sites by apurinic/apyrimidinic endonuclease [23].…”

“…In bacteria such as Escherichia coli and Salmonella typhimurium, dUTP biosynthesis is an obligatory intermediate in the de novo synthesis of dTTP [3][4][5]. Since DNA polymerases can utilize dUTP in place of dTTP [6,7], some incorporation of dUMP into the bacterial chromosome is unavoidable.…”

“…Mutations in the E. coli dUTPase gene (dut) were shown to cause a large increase in dUMP incorporation into the genome [3]. Subsequently, it was found that while some dut mutations were lethal, their lethality could be suppressed by null mutations in the dcd gene which, presumably, reduced the accumulation of dUTP and, hence, uracil in DNA [10].…”

Structure of the gene for human uracil—DNA glycosylase and analysis of the promoter function

“…The successive steps in the base excision repair pathway are thought to involve action of an AP endonuclease, DNA deoxyribophosphodiesterase, a DNA polymerase and a DNA ligase [1,2]. Uracil in DNA results from deamination of cytosine [3] or incorporation of dUMP instead of dTMP [4,5]. Deamination will result in a GC to AT transition mutation unless uracil is removed before the next round of replication.…”