5-Fluorouracil Is Efficiently Removed From DNA by the Base Excision and Mismatch Repair Systems  Franziska Fischer, Katja Baerenfaller, Josef Jiricny  Gastroenterology  Volume 133, Issue 6, Pages 1858-1868 (December 2007) DOI: 10.1053/j.gastro.2007.09.003 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Chemical structures of halogenated pyrimidines and the recognition of FU in DNA by the human mismatch binding factor MutSα. (A) Keto-enol tautomerism of uracils substituted with fluorine (FU), bromine (BrU), and iodine (IU). The question mark indicates the uncertainty regarding the prevalence of the enol form of FU. (B) Binding of MutSα to FU/G and FU/A oligonucleotide substrates. The 5′-[32P]-labeled oligonucleotides (A/T, G/T, FU/A, FU/G) were incubated with 40 nmoles (lanes 1–4), 70 nmoles (lanes 5–8), or 100 nmoles (lanes 9–12) of purified human MutSα. The figure is an autoradiogram of a 5% native polyacrylamide gel. Gastroenterology 2007 133, 1858-1868DOI: (10.1053/j.gastro.2007.09.003) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Processing of FU-containing base pairs in nuclear extracts of human cells. (A) Schematic representation of the construction of the FU-containing DNA substrates and of the in vitro MMR assay. (B) Susceptibility of FU-containing heteroduplex substrates to cleavage with AclI. The FU/A (lane 1) substrate was cleaved at position 46 (see panel A above), whereas the FU/G (lane 2) and G/T (lane 3) substrates were refractory to cleavage at this position. (C) In vitro MMR assays performed with the FU/A (lanes 1 and 2), FU/G (lanes 3 and 4), or G/T (lanes 5 and 6) substrates. The 1516-bp and 1307-bp restriction fragments are indicative of repair (for details see Figure 2A). The reactions were carried out in nuclear extracts of MLH1-proficient (+) or MLH1-deficient (−) 293T-Lα cells. Gastroenterology 2007 133, 1858-1868DOI: (10.1053/j.gastro.2007.09.003) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Contribution of different glycosylases to the repair of FU/G mispairs. (A) Mismatch repair assay in the presence of different concentrations of uracil-DNA glycosylase inhibitor (UGI). The reactions were performed in nuclear extracts of MLH1-proficient (+) or MLH1-deficient (−) 293T-Lα cells, with the nicked FU/G (lanes 1–5) or G/T (lane 6) substrates. The used UGI concentrations (U) are indicated in units defined by the manufacturer. (B) Effect of MBD4 depletion on FU/G repair. Efficiency of MBD4 immunodepletion from 293T-Lα nuclear extracts was confirmed by Western blot analysis (MBD4−; left panel, lanes 1 and 2). The MMR assays (right panel) were performed with undepleted (lanes 1 and 2) or MBD4-depleted nuclear extract (lanes 3–6). The DNA bands representing the repaired fragments were quantified using ImageQuant TL, Amersham Biosciences (right bottom panel). (C) Effect of TDG depletion on FU/G repair. The reactions are identical to those shown in panel B, except that both SUMOylated and unmodified forms of TDG (arrows) were immunodepleted. Gastroenterology 2007 133, 1858-1868DOI: (10.1053/j.gastro.2007.09.003) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Lack of TDG, UNG and MMR abolishes FU/G repair in vitro. (A) Western blot of MMR-deficient (lanes 1 and 3) and -proficient (lanes 2 and 4) extracts of 293T-Lα cells immunodepleted of TDG (lanes 1 and 2). (B) The MMR assays (upper panel) were carried out either with undepleted extracts (lanes 1 and 2), or with extracts immunodepleted of TDG (lanes 3-6). In assays pictured in lanes 2-6, UNG was inhibited by Ugi. The relative efficiencies of the MMR reactions were quantified by PhosporImager (lower panel). Gastroenterology 2007 133, 1858-1868DOI: (10.1053/j.gastro.2007.09.003) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 FU/A repair in human cell extracts. (A) Western blot of MMR-deficient (lanes 1 and 3) and -proficient (lanes 2 and 4) extracts of 293T-Lα cells immunodepleted of TDG (lanes 1 and 2). (B) The MMR assays (upper panel) were carried out either with undepleted MMR-deficient (lane 1) or -proficient (lane 2) extracts, or with MMR-deficient (lanes 3 and 5) or -proficient (lanes 4 and 6) extracts immunodepleted of TDG, in which UNG was inhibited by Ugi. The incorporation of [α-32P]AMP into the repair intermediates and products was quantified by PhosporImager (lower panel). See text for details. Gastroenterology 2007 133, 1858-1868DOI: (10.1053/j.gastro.2007.09.003) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Processing of FU-containing substrates by SMUG1. (A) SMUG1 is present in the extracts of 293T-Lα cells in large amounts, as shown by Western blot analysis. LoVo extracts, which express lower amounts of SMUG1 messenger RNA than 293T-Lα cells as judged by microarray experiments (J. Sabates-Bellver, unpublished observations), contain correspondingly lower amounts of the protein. (B) In vitro activity of SMUG1 on FU-containing oligonucleotides. The FU/A and FU/G substrates were incubated either under MMR conditions (10 × MMR buffer, lanes 1–4) or in buffer recommended by the manufacturer (10 × DDR buffer, lanes 5–10, see Materials and Methods section) in the presence (+) or absence (−) of MLH1-deficient nuclear extract of 293T-Lα cells (NE MLH1−). As shown, the recombinant enzyme is inactive in the MMR buffer, which might explain the absence of processing of FU-containing substrates in the cell extracts. The figure is an autoradiograph of a 12% denaturing polyacrylamide gel. Gastroenterology 2007 133, 1858-1868DOI: (10.1053/j.gastro.2007.09.003) Copyright © 2007 AGA Institute Terms and Conditions