Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

Abstract

DNA polymerase III mis-insertion may, where not corrected by its 3'→ 5' exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The k _cat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo⁸G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The K _M values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N ⁴- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

Keywords: DNA base mismatch; DNA glycosylase; Escherichia coli Fpg; base excision repair; cytosine:cytosine mismatch; mutM; thymine:thymine mismatch.

FIGURE 1

Escherichia coli Fpg protein incises at unmethylated and methylated cytosine when placed opposite C and T in DNA. (A) Schematic representation of DNA substrates. Fluorescently labeled DNA oligonucleotides (*, phosphodiester bonds protected by phosphorothioate) had a spectrum of different studied bases at one site, as indicated. The upper strand was primarily labeled, its variable base defined by X (indicated in red); the lower strand was occasionally labeled, its variable base defined by Y (indicated in blue). The color code is kept throughout all the subsequent figures and tables indicating which variables in which strand were tested in a particular experiment. A variable base in the labeled strand (the assessed one) is always written as the first in a base pair. See Supplementary Figure 1 for an outline of the assay. (B) Activity for C opposite all major bases. (C) Activity for methylated Cs opposite C. (D) Activity for methylated Cs opposite T. In panels (B–D), DNA (upper strand labeled) X substrate [see panel (A), 1 pmol] was incubated alone (lanes 1–4) or with Fpg (13 pmol; lanes 5–8) at 37°C in NEB1 buffer (10 mM Bis–Tris-propane-HCl, pH 7.0, 10 mM MgCl₂), 1 mM DTT, 0.1 mg/mL BSA for 1 h. U⋅G-DNA (30 nt; 1 pmol) was incubated without (lane 9) or with Ung (1.95 pmol; lane 10) followed by NaOH/heat treatment, and was used as a negative and positive control, respectively, for active Ung, and to convert U⋅G-DNA into AP-DNA to demonstrate active Fpg (i.e., AP lyase activity; lane 11). (E) Activity for C and m⁵C opposite C, when the lower strand with the Y variable was labeled. DNA substrate (1 pmol) was incubated alone (lanes 1 and 2) or with Fpg (lanes 3 and 4) using the controls (lanes 5–7) described above. (F) Activity for C, m⁵C, or oxo⁸G opposite C compared to the other homo-mismatches. (G) Activity for C opposite C compared to Tg and dHT opposite A. In panels (F,G), DNA (upper strand labeled) X substrate [see panel (A), 1 pmol] was incubated alone (lanes 1–6 and 1–3, respectively) or with Fpg (lanes 7–12 and 4–6, respectively) using the controls (lanes 13–15 and 7–9, respectively) described above. (H) The percent of the labeled strand incised at unmethylated and methylated C opposite C. (I) The percent of the labeled strand incised at unmethylated C opposite T, A, or G compared to methylated C opposite T. (J) The percent of the labeled strand incised at most homo-mismatches compared to oxidized bases in DNA. These column graphs show the average values (±SD) obtained from 4 to 10 independent experiments as presented in panels (B–G), the first base in each pair being the one assayed.

FIGURE 2

Schiff base trapping analysis of Fpg protein. DNA substrate (1 pmol) with either X⋅C (left panel) or X⋅G (right panel) base pair, alone as a negative control (lanes 1 and 2), or together with Fpg (10 pmol; lanes 3 and 4), was incubated with 50 mM NaBH₄ in reaction buffer at 37°C for 1 h (final volume, 10 μL). U⋅G-DNA (30 nt; 1 pmol) incubated alone (lane 5), with Ung (10 pmol; lane 6), or with Ung and Fpg (10 pmol each; lane 7), was used as a negative and positive control for active Ung and Fpg, respectively, Ung converting U⋅G-DNA into AP-DNA to be trapped by Fpg. The trapped protein was separated from un-trapped protein by denaturing PAGE. The experiments were performed 10 (X⋅C) or 5 times (X⋅G), the result being the same.

FIGURE 3

Definition and processing of the 3′-end after Fpg-mediated incision of m^N4,5C⋅C-DNA. DNA substrate (Figure 1A; 1 pmol) was incubated without (lane 1) or with Fpg (13 pmol; lanes 2–4) at 37°C for 30 min, followed by no addition (lanes 1 and 2), addition of 0.083 pmol endonuclease IV (Nfo; lane 3) or addition of 0.29 pmol T4 polynucleotide kinase (PseT; lane 4), and incubation for an additional 30 min (final volume, 10 μL). Incised DNA was separated from un-incised DNA by denaturing PAGE (Supplementary Figure 1) at 500 V for 4 h. Abbreviation: 3′-P, 3′-phosphate. The 5′-labeled strand is indicated by 5′ in magenta.