Fuchs RP , Isogawa A , Paulo JA , Onizuka K , Takahashi T , Amunugama R , Duxin JP , Fujii S .

R01 GM132129 NIGMS NIH HHS , JP20H05392 Japan Society for the Promotion of Science , JP20H03186 Japan Society for the Promotion of Science , R01 GM132129 NIH HHS

             DNA repair
             mismatch repair

	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 2 DNA repair synthesis in alkylated and undamaged control plasmid DNA in NPE. (A) Outline of the spot assay. Plasmids were incubated in nuclear extracts supplemented with α32P-dATP; at various time points, an aliquot of the reaction mixture was spotted on DEAE paper (see 'Materials and methods'). The dot blot is shown for the sake of illustration only. (B) Plasmid DNA pBR322 (4.3 kb) samples, modified to a similar extent with -MMS, -MNU and -ENU, were incubated in nucleoplasmic extracts (NPE) supplemented with α32P-dATP at room temperature; incorporation of radioactivity was monitored as a function of time using the spot assay described above (A). Undamaged plasmid DNA0 was used as a control. At each time point, the average values and standard deviation from three independent experiments were plotted. The y-axis represents DNA repair synthesis expressed as a fraction of input plasmid replication (i.e., 10% means that the observed extent of repair synthesis is equivalent to 10% of input plasmid replication). This value was determined knowing the average concentration of dATP in the extract (50 μM) and the amount of added α32P-dATP. (C) N-methyl-N-nitrosourea (MMS)- and N-methyl-N-nitrosourea (MNU)-treated plasmids were incubated in NPE, supplemented or not, by aphidicolin (150 μM final). After 1 hr of incubation, plasmids were purified and analyzed by agarose gel electrophoresis under neutral loading conditions. The gel was imaged by fluorescence (left: ethidium bromide image) and by autoradiography (right: 32P image). The number below each lane indicates the total amount of signals per lane (expressed in arbitrary units [AU]). Aphidicolin treatment decreases incorporation into MNU-treated plasmid close to fourfold, while it affected incorporation into MMS-treated plasmid only 1.6-fold. (D) Samples as in (C). Gel loading is performed under alkaline conditions to denature DNA before entering the neutral agarose gel, allowing single-stranded nicks present in DNA to be revealed. The number below each lane indicates the amount of signals per lane (AU).
	Figure 3 Stimulation of MMR at a single O6mG site by N-alkyl adducts in cis. (A) Covalently closed circular (ccc) plasmids (pAS200.2, 2.1 kb) containing a site-specific O6mG:C base pair (plasmid mGC) and the corresponding lesion-free control (plasmid GC) were constructed (Isogawa et al., 2020). Similarly, plasmids with a site-specific GT or a O6mG:T mismatch were constructed. All the four constructs were treated with methyl-methane sulfonate (MMS) in order to introduce random N-alkyl (7mG and 3mA) adducts, generating plasmids GC+MMS, mGC+MMS, GT+MMS, and mGT+MMS. We adjusted the MMS reaction conditions as to introduce ≈ nine adducts per plasmid (i.e., one N-alkylation adduct every ≈500 nt). The resulting proportion of O-alk and N-alkyl adducts mimics the proportion in N-methyl-N-nitrosourea (MNU)-treated plasmids. The single O6mG adduct and the randomly located N-alkyl adducts are represented by a star and red dots, respectively. (B) Plasmids described above were incubated in nucleoplasmic extracts (NPE) supplemented with α32P-dATP at room temperature; incorporation of radioactivity was monitored as a function of time using the spot assay. The y-axis represents the percentage of DNA repair synthesis with respect to input DNA (i.e., 10% means that the observed extent of repair synthesis is equivalent to 10% of input plasmid replication). Overall, incorporation into GT and mGT plasmids is higher than incorporation into their GC and mGC counterparts. Incorporation attributable to repair at the O6mG:C lesion is increased close to threefold due to the presence of random N-alkyl lesions introduced by MMS treatment. The stimulatory effect of random N-alkyl lesions on GT and mGT repair is observed but is slightly less pronounced than for mGC. (C) The same plasmids were incubated for 2 hr in NPE, purified, resolved by agarose gel electrophoresis, and revealed by ethidium bromide fluorescence and 32P autoradiography. The total amount of signals per lane is indicated (arbitrary units [AU]). As expected, the amount of plasmid extracted from each incubation mix is relatively constant, as quantified below the ethidium bromide image. Increase in repair at the O6mG:C lesion due to MMS treatment (2.8-fold) is in good agreement with data in (B).
	Figure 4 Double-strand breaks occur in MNU-treated plasmids during incubation in extracts. (A) Analysis by agarose gel electrophoresis (AGE) of alkylated plasmids (pEL97: 11.3 kb) incubated in nucleoplasmic extracts (NPE) in the presence of α32P-dATP. Plasmid pEL97 was treated with methyl-methane sulfonate (MMS), N-methyl-N-nitrosourea (MNU)+, and ENU as to introduce ≈ one alkylation event, on average, every 500 nt. For MNU, a plasmid with twice the level of alkylation (MNU++, one lesion every 250 nt) was also produced (Figure 4—figure supplement 1). Alkylation of these plasmids essentially not affected their migration on agarose gels (Figure 4—figure supplement 2A). After 2 hr of incubation, the reaction was stopped and a known amount of pBR322 (10 ng) plasmid was added as an internal standard. Ethidium bromide image: in different lanes, the internal standard band, pBR (covalently closed circular [ccc]), appears to be of similar intensity (1158 +/- 95 arbitrary units [AU]), assessing reproducible DNA extraction. For the alkylated plasmids, incubation in NPE led to massive conversion from ccc to relaxed plasmids. 32P image: little incorporation of 32P-dATP is seen in DNA0 and in MMS-treated plasmids compared to MNU- and ENU-treated plasmids as shown by the relative incorporation levels normalized to one for untreated plasmid (DNA0). As expected, the MNU++ sample exhibits about twice the amount of incorporated radioactivity compared to MNU+. In both ethidium bromide and 32P images, a small amount of linear plasmid is seen mostly in the MNU++ sample. This band is also visible in the MNU+ and ENU lanes although at a weaker intensity. (B) Quadratic dose-response for double-strand break (DSB) formation. When the % of linear form (linear/(linear + oc)) is plotted as a function of the square dose of MNU (mM2) for untreated, MNU+, and MNU++ plasmids, we observed a straight line (y = 1.4173x - 0.0288; R² = 0.9999).
	Figure 5 Simultaneous repair of two closely spaced MNU-induced lesions may lead to a DSB. Such a situation occurs when an N-alkyl lesion located within ≈500 nt of an O6mG lesion is processed simultaneously (‘Lesion Arrangement at-risk’). Note that the mismatch repair (MMR) excision track can occur on either strand as described for noncanonical MMR (Peña-Diaz et al., 2012). Reaction of N-methyl-N-nitrosourea (MNU) with double-stranded DNA induces N-alkylation adducts, mostly 7mG and 3mA shown as * and O-alkylation adducts (O6mG), at a ratio of 10:1 approximately. Step 1: a base excision repair (BER) event is initiated at an N-alkyl adduct, creating a nick. Step 2: concomitantly, an MMR event takes place, in the opposite strand, at a nearby O6mG:C site. Step 3: the MMR machinery extends the nick into a several hundred nt-long gap by means of Exo1 action. Step 4: the two independently initiated repair events lead to a double-strand break (DSB), if the MMR gap reaches the BER-initiated nick before resealing.

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