Xue Z et al. (2025), Pathways and products of base excision DNA repa...

XB-ART-61646

Nucleic Acids Res 2025 Nov 26;5322:. doi: 10.1093/nar/gkaf1326.

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Pathways and products of base excision DNA repair in Xenopus laevis eggs: contrast with human cell pathways.

Xue Z , Sutton PJ , Haley JD , Brownlee CW , Demple B .

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Endogenous DNA damage is mutagenic and cytotoxic unless removed by base excision DNA repair (BER). BER replaces 2-10 nucleotides in "long patch" (LP-BER) or just one nucleotide in the simpler "short-patch" SP-BER. LP-BER is required for some oxidative lesions. Xenopus laevis egg extracts (HSS) had robust BER of diverse lesions (uracil and various abasic sites). It was surprising to find no evidence for HSS repair of 8-oxoguanine, an important mutagenic lesion. Also unexpected was the independence of HSS BER from DNA polymerase β (Polβ), which typically contributes both DNA repair synthesis and 5' "trimming" to SP-BER. Instead, HSS BER was blocked by aphidicolin inhibiting the replication DNA polymerases δ and ε. Added human Polβ restored full repair, requiring only its 5'-trimming activity, not its DNA polymerase function. Mass-labeled probes revealed that Xenopus BER repair patches were 80%-90% 2-nucleotide (2-nt) LP-BER tracts. Supplementing the low Polβ level in HSS with recombinant enzyme shifted BER toward 1-nt (SP-BER) patches, also influenced by the surrounding sequence. Excision upstream (5') of the lesion occurred in most of the frog BER products. In contrast, human cell (HEK293) extracts had Polβ-dependent BER, with mostly 2-nt repair patches. Human LP-BER was driven by the Fen1 flap endonuclease, but it did not include 5' excision.

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	Graphical Abstract.
	Figure 1. Xenopus HSS has robust BER activity. (A) The U:A substrate was incubated with Xenopus HSS for the indicated times, then digested with UDG + Ape1 to nick the unrepaired portion. Full-length substrate (indicated by C above lane 1), UDG + Ape1 digested substrate (lane 2), and UDG + Ape1 digested, HSS-repaired samples (lanes 3–6) were electrophoresed in a denaturing polyacrylamide gel, then imaged using a fluorescent imager. HSS was pre-incubated with uracil glycosylase inhibitor (UGI) to inhibit endogenous UDG activity before adding the U:A substrate (B) or the U:G substrate. (C) The F:A substrate was incubated with Xenopus HSS for the indicated times, without (D) or with (E) added UGI (0.5, 1, 2, 3 units/μl). Ape1 was added where indicated to cleave unrepaired molecules. (F) The LP-BER substrate rAP was incubated with Xenopus HSS. Nth was used as a control for AP site reduction since Nth cannot digest rAP, while Ape1 can. For all panels, C indicates the starting substrate, P the repaired product, and U the unrepaired substrate or a repair intermediate. F, tetrahydrofuran; rAP, reduced AP site.
	Figure 2. Repair rates, base pairing, and contributions of DNA repair polymerases in the HSS. (A–C) Linear duplex oligonucleotide substrates were incubated with Xenopus HSS for the indicated times, and the repaired products were resolved by denaturing polyacrylamide gel electrophoresis as in Fig. 1, with the fraction repaired was quantified by ImageJ. Each data point resulted from three biological replicates, and the data shown are the mean ± standard deviation (SD). (A), U paired with A or G; (B), F paired with A, C, or G; (C), reduced AP site (rAP) paired with A, C, or G. n = 3 biological replicates, and the data are means ± SD. (D, E) Aphidicolin sensitivity of BER in Xenopus HSS. Where indicated, the HSS was pre-incubated with aphidicolin (100 μg/ml) to inhibit replicative DNA polymerases, with or without added Polβ, incubated with the substrate, and then analyzed for repair as in Fig. 1. (D), U:A substrate, with UDG + Ape1 to cleave unrepaired molecules. (E), F:A substrate, with Ape1 to cleave unrepaired molecules. (F, G) Effect of added ddTTP on the repair reactions. (F) Repair of the U:A substrate; (G), repair of the F:A substrate. C, control; P, repaired product; U, unrepaired substrate or repair intermediate.
	Figure 3. The isotopic labeling plasmid approach to determine BER patch size. (A) The target DNA fragment for “oligo swapping” was inserted into the multicloning site of pUC19, and one of the Nt.BspQI sites was deleted. (B) The DNA sequence flanking the target site, indicating the cut sites of key restriction enzymes. The incoming biotin-labeled synthetic oligonucleotide is shown above the target sequence; the G indicates isotopically labeled dGMP residues (fully substituted with 13C and 15N). The segment highlighted in green is used for mass spectrometry analysis. (C) Overview of the oligo swapping procedure (see the “Materials and methods” section for details). (D) Stepwise products from the substrate synthesis procedure in panel (C) were loaded on an ethidium bromide-containing agarose gel (lanes 2–5) and subjected to electrophoresis. A DNA ladder was loaded in lane 1. To determine the oligo swapping efficiency, the plasmid substrate was digested with UDG and NdeI (lane 6): removal of U prevents cleavage by NdeI. (E) Mass spectrometry analysis confirming the target fragment released from plasmid substrate by NdeI and HaeII. (F) A sample ethidium bromide-containing agarose gel image showing the repair product of plasmid substrate by Xenopus HSS at the indicated time points, with the repaired substrate susceptible to UDG and NdeI cleavage (lanes 6–9). A DNA ladder was loaded in lane 1. Native pUC19 was digested with PstI (lane 2) or NdeI (lane 3) for size markers. Plasmid substrate was digested with PstI (lane 4) or UDG + NdeI (lane 5) to confirm the presence of uracil. P, repaired product; U, unrepaired substrate. (G) Repair rates for U:A and rAP:A substrates incubated with Xenopus HSS. Gel images were quantified using ImageJ. P < .05. n = 3 biological replicates and the data are means ± SD.
	Figure 4. DNA polymerase dictates BER patch size. (A) The BER patch size for a U:A plasmid substrate generated by Xenopus HSS, or HSS supplemented with the indicated proteins and inhibitors. Aph, aphidicolin (100 μg/ml). n = 3 independent experiments and the data are means ± SD. * indicates P < .001, ** indicates P < .0001. (B) Effect of ddTTP in the presence of added Polβ or Polλ. Incubation time was 60 min. Treatment with UDG and Ape1, followed by electrophoresis on a denaturing polyacrylamide gel, revealed fully repaired (P) and unrepaired (U) molecules. (C) Effect of Polβ polymerase and dRp lyase activities on BER patch size in the Xenopus HSS.
	Figure 5. Effect of downstream sequence and topological state on BER patch size. Various lesions in (A) relaxed N3–1 V1 plasmid substrate, (B) supercoiled N3–1 V1 plasmid substrate, (C) relaxed N3–1 V7 plasmid substrate, and (D) supercoiled N3–1 V7 plasmid substrate were tested. Polβ final concentration was 50 nM. n = 3 independent experiments, and the data are means ± SD.
	Figure 6. Upstream (5′) excision is a common event in Xenopus HSS BER. 5′ excision gap size of relaxed (A) and supercoiled (B) N3–1 V7 plasmid substrate with the indicated lesions. Polβ final concentration was 50 nM. (C) Dead-end products generated by Polβ and Polλ in the presence of ddTTP. n = 3 independent experiments and the data are means ± SD. The F:A oligonucleotide substrate was incubated with Xenopus HSS supplemented with 50 nM Polβ or Polλ in reaction buffers containing 0%, 50%, or 100% ddTTP (replacing dTTP) for the indicated times. The products were digested with Ape1 and loaded on a denaturing polyacrylamide gel (lanes 6–23). Full-length substrate (lane 1) and digested substrate (lane 2) were included as markers. Lanes 3–5, repair of F:A by Xenopus HSS without any supplementation.
	Figure 7. Polβ actively participated in the BER of G1/S synchronized and asynchronous HEK293 FT WCE. The U:A (A) or F:A (B) oligo substrate was incubated with asynchronous HEK293 FT WCE supplemented with aphidicolin (100 μg/ml) or reaction buffers containing 50% or 100% ddTTP for 1 h. The ratio of repaired products was quantified by ImageJ. Cell cycle profiles of asynchronous (C) and double thymidine synchronized (D) HEK293 FT cells. The sensitivity of WCE made from synchronized HEK 293 FT cells to aphidicolin (100 μg/ml) and ddTTP was determined using the U:A (E) or F:A (F) oligo substrate as in panels (A) and (B). n = 3 independent experiments and the data are means ± SD. * indicates P < .001. ** indicates P < .0001. Aph, aphidicolin. Async, asynchronous.
	Figure 8. hFen1 immunodepletion increased SP-BER product. (A) The BER patch size for a U:A and rAP:A plasmid substrate generated by asynchronous HEK 293 FT WCE or WCE supplemented with Polβ (50 nM) and aphidicolin (100 μg/ml). Aph, aphidicolin. ** indicates P < .0001. n = 3 independent experiments and the data are means ± SD. Async, asynchronous. (B) The hFen1 abundance in asynchronous HEK 293 FT WCE and anti-hFen1 antibody immunodepleted WCE were determined using western blot. ID: Immunodepletion. (C) The BER patch size for a U:A plasmid substrate generated by asynchronous HEK 293 FT WCE, hFen1 immunodepleted WCE, hFen1 (150 nM) supplemented hFen1 immunodepleted WCE, and xFen1 (150 nM) supplemented hFen1 immunodepleted WCE. ** indicates P < .0001. ns, not significant. n = 3 independent experiments and the data are means ± SD.
	Figure 9. The effect of protein concentration, substrate topological state, and sequence context on BER patch size distribution on HEK 293 WCE. (A) The BER patch size generated by the indicated asynchronous HEK 293 FT WCE concentrations is shown for the N3–1 V1 U:A plasmid substrate. (B) Various lesions in relaxed or supercoiled N3–1 V1 plasmid substrate. (C) The BER patch size generated by asynchronous HEK 293 FT WCE is shown for the N3–1 V7 U:A plasmid substrate (C), N3–1 V6 U:A/U:G plasmid substrate (D) and N3–1 V6 U:A/U:G plasmid substrate. (E) n = 3 independent experiments, and the data are means ± SD. * indicates P < .05. **** indicates P < .0001. (F) A sample mass spectrometry result from asynchronous HEK 293 FT WCE incubated with N3-1 V7 U:A plasmid substrate. 0 nt, 1 nt, 2 nt, 3 nt, and 4 nt indicate the repair products that went through 0 nt, 1 nt, 2 nt, 3 nt, and 4 nt + longer 5′ excision.