Figure 1. Pull-down of proteins that bind to alkylated versus untreated plasmid DNA.

(A) Experimental workflow. Plasmid DNA (pAS04, 6.5 kb) was treated with alkylating agents under conditions leading to a similar extent of N-alkylation (≈ one alkaline cleavage site every 500 nt) (Figure 1—figure supplement 1A). Immobilized plasmid DNA was incubated in Xenopus nucleoplasmic extracts (NPE) for 10 min at room temperature under mild agitation. The reaction was stopped by addition of formaldehyde (0.8% final) to cross-link the protein-DNA complexes. The beads were processed and analyzed by polyacrylamide gel electrophoresis (PAGE) or by mass spectrometry (MS) as described in 'Materials and methods'. (B) Relative abundance of proteins captured on N-methyl-N-nitrosourea (MNU)-treated versus -untreated DNA0. Proteins captured on equal amounts of MNU-treated or -untreated plasmid were analyzed by label-free MS in triplicate. For all proteins, average spectral count values in the MNU-treated plasmid sample were divided by the average spectral count values in the DNA0 sample. The resulting ratio is plotted as its log₂ value along x-axis. The statistical significance of the data is estimated by the p-value in the Student’s t-test and plotted as -log₁₀p along y-axis. Proteins enriched on MNU versus untreated plasmid DNA appear on the right-side top corner and essentially turn out to be mismatch repair (MMR) proteins labeled in red (B). Data shown are analyzed using Xenbase database. (C) Relative abundance of proteins captured on methyl-methane sulfonate (MMS)-treated versus -untreated DNA0. Proteins captured on equal amounts of MMS-treated or -untreated plasmid were analyzed by label-free MS in triplicate. The data are analyzed and plotted as in panel (B) for MNU using Xenbase database. Proteins (labeled in green in B and C) are found enriched or excluded in both MMS versus DNA0 and MNU versus DNA0 plasmids. We suggest these proteins are recruited or excluded from binding to DNA by the abundant class of N-alkylation adducts that both MMS- and MNU-treated plasmids share in common (~27 N-alkyl adducts per plasmid).

Figure 1—figure supplement 1. Alkylation reaction conditions and differential protein capture data.

( A) Alkylation reaction conditions and alkaline cleavage. The alkylation reaction conditions (concentration, reaction temperature, and time) were adjusted by successive trials until an average of one N-alkyl adduct per 500 nt was reached. The desired level of alkylation (≈1 N-alkyl adduct/500nt) was attested using the alkaline cleavage procedure, followed by neutral agarose gel electrophoresis (for details, see 'Materials and methods'). (B) Polyacrylamide gel electrophoresis (PAGE) analysis and silver staining of the captured proteins. Proteins captured on equal amounts of immobilized plasmid DNA incubated in nucleoplasmic extracts (NPE) (as described in Figure 1A) were resolved by denaturing gel electrophoresis and silver stained. Samples, corresponding to ≈30 ng of immobilized input plasmid DNA, were loaded on a 4–15% PAGE (Biorad pre-cast) gel, run at 200 volts for 32 min, and silver stained. Lanes DNA0, MMS, MNU, and ENU display a complex pattern of proteins (total amount of proteins per lane estimated at 100–200 ng). To monitor non-specific protein binding to beads, we included a negative control containing the same amount of M280 beads in the absence of plasmid DNA (noDNA). Importantly, in the noDNA lane, only a low amount of residual protein is visible, indicating efficient removal of non-specifically bound proteins during the washing procedure. (C) Relative abundance of proteins captured on N-methyl-N-nitrosourea (MNU)- versus methyl-methane sulfonate (MMS)-treated plasmids. Proteins captured on equal amounts of MNU- or MMS-treated plasmids were analyzed by label-free mass spectrometry (MS) in triplicate. The data are analyzed and plotted as described in Figure 1B using Xenbase database. Proteins enriched on MNU- versus MMS-treated DNA appear on the right-side top corner and essentially turn out to be mismatch repair (MMR) proteins labeled in red (Figure 1B).