Uracil in DNA: consequences for carcinogenesis and chemotherapy

Abstract

The synthesis of thymidylate (TMP) occupies a convergence of two critical metabolic pathways: folate metabolism and pyrimidine biosynthesis. Thymidylate is formed from deoxyuridylate (dUMP) using N(5),N(10)-methylene tetrahydrofolate. The metabolic relationship between dUMP, TMP, and folate has been the subject of cancer research from prevention to chemotherapy. Thymidylate stress is induced by nutritional deficiency of folic acid, defects in folate metabolism, and by antifolate and fluoropyrimidine chemotherapeutics. Both classes of chemotherapeutics remain mainstay treatments against solid tumors. Because of the close relationship between dUMP and TMP, thymidylate stress is associated with increased incorporation of uracil into DNA. Genomic uracil is removed by uracil DNA glycosylases of base excision repair (BER). Unfortunately, BER is apparently problematic during thymidylate stress. Because BER requires a DNA resynthesis step, elevated dUTP causes reintroduction of genomic uracil. BER strand break intermediates are clastogenic if not repaired. Thus, BER during thymidylate stress appears to cause genome instability, yet might also contribute to the mechanism of action for antifolates and fluoropyrimidines. However, the precise roles of BER and its components during thymidylate stress remain unclear. In particular, links between BER and downstream events remain poorly defined, including damage signaling pathways and homologous recombination (HR). Evidence is growing that HR responds to persistent BER strand break intermediates and DNA damage signaling pathways mediate cross talk between BER and HR. Examination of crosstalk among BER, HR, and damage signaling may shed light on decades of investigation and provide insight for development of novel chemopreventive and chemotherapeutic approaches.

Figure 1. Simplified schematic of folate metabolism focused on the synthesis and utilization of N⁵,N¹⁰ methylene tetrahydrofolate

N⁵,N¹⁰ Methylene tetrahydrofolate can be utilized as the methyl donor and reductant in TMP synthesis, the methyl donor for the conversion of homocysteine to methionine, and the eventual formyl donor for de novo purine synthesis. Enzymes are in italics. MTHFR, methylene-THF reductase; SHMT, serine hydroxymethyl transferase; TS, thymidylate synthase; MTHFD, methylene-THF dehydrogenase; SAM, S-adenosyl methionine; Met, methionine; H-Cys, homocysteine; THF, tetrahydrofolate; DHF, dihdyrofolate.

Figure 2. Simplified schematic of dUMP and TMP metabolism leading to DNA synthesis

Enzymes are in italics. TS, thymidylate synthase; TK, thymidine kinase; TMPK, thymidylate kinase, dUTPase, dUTP nucleotidohydrolase. RNR, ribonucleotide reductase; dCMP-DA, dCMP deaminase. TS is the de novo source of TMP, while TK provides a salvage route to TMP and dUMP. dUTPase prevents dUTP accumulation. Raltitrexed and pemetrexed are folate analogs that interfere with binding of N⁵,N¹⁰ Methylene tetrahydrofolate to TS. Fluorodeoxyuridylate (FdUMP) binds in the nucleotide binding pocket and forms an irreversible ternary complex.

Figure 3. Simplified schematic of base excision repair during TLS

In the first step of BER, UDG activity catalyzes uracil release and produces an abasic site in DNA. AP endonuclease produces a nick 5′ to the abasic site. The dRP lyase activity of pol β creates a 5′-phosphate and polymerase activity fills the one nucleotide gap in short-patch BER. DNA ligase seals the nick. During TLS, insufficient TS activity decreases TTP and increases dUTP, overwhelming dUTPase and driving genomic uracil incorporation.

Figure 4. Simplified schematic of the links between TLS and homologous recombination repair

DNA double strand breaks generated from persistent BER strand break intermediates and/or stalled/collapsed replication forks are thought to be processed by HR. The simplified biochemical steps necessary for HR include search for homology, strand invasion, DNA synthesis, and an endonuclease to resolve the Holliday junction. The outcome of HR could potentially be detectable as a crossover event resulting in sister chromatid exchanges or gene conversion events. If left unrepaired, double strand breaks lead to non-viable cells or chromosomal rearrangements detectable as translocations in a population of surviving cells.