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We performed repair reactions with DNA substrates containing either a U:G or a U:A pair, in the presence of a single deoxynucleotide (dATP, dCTP, dGTP or dGTP) (Figure 5). All steps were performed at 0–4°C. The full text of this article hosted at iucr.org is unavailable due to technical difficulties. Therefore, it probably represents a 5′‐P terminus. Where indicated, DNA was resuspended in 5 μl of SuRE/Cut Buffer L containing 5 U of HpaII (Roche) and incubated at 37°C for 1 h. Reactions were then stopped by adding 5 μl of 90% formamide and then heating at 95°C for 5 min. Thus, AP endonucleases and AP lyases generate 5′‐ and 3′‐blocked ends, respectively. Resolving these and other questions will require experimental data on different DNA lesions, and the identification of enzymes specifically involved either in SP or LP BER. As experimental data accumulate for non‐mammalian systems, it is becoming clear that there are significant differences in the strategies employed by different species during the post‐excision events that take place in BER (Kelley et al., 2003). . This avoids the need for an AP endonuclease to clear the 3′‐blocked end, but implies that 3′‐phosphoesterase activity may be required to restore conventional 3′‐OH. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. Size markers corresponding to 28, 29 and 30 nt were loaded on lane 7. Therefore, it probably represents a 5′‐dRP blocked terminus. This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways. When analyzing 5′ ends, reaction products were stabilized by the addition of freshly prepared sodium borohydride (NaBH4; Sigma‐Aldrich) to a final concentration of 300 mm, incubation at 0°C for 30 min and desalting in a microspin G‐25 column (GE Healthcare, http://www.gelifesciences.com). Reaction products were separated in a 12% denaturing polyacrylamide sequencing gel (40 × 20 cm) containing 7 m urea. Enter your email address below and we will send you your username, If the address matches an existing account you will receive an email with instructions to retrieve your username, I have read and accept the Wiley Online Library Terms and Conditions of Use, Role of the Arabidopsis DNA glycosylase/lyase ROS1 in active DNA demethylation, Mitochondrial base excision repair of uracil and AP sites takes place by single‐nucleotide insertion and long‐patch DNA synthesis, Poly(ADP‐ribose) polymerase in base excision repair: always engaged, but not essential for DNA damage processing, Orchestration of base excision repair by controlling the rates of enzymatic activities, A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in, Higher plants possess two structurally different poly(ADP‐ribose) polymerases, Fidelity of uracil‐initiated base excision DNA repair in DNA polymerase β‐proficient and ‐deficient mouse embryonic fibroblast cell extracts, Abasic sites in DNA: repair and biological consequences in Saccharomyces cerevisiae, DNA polymerase λ mediates a back‐up base excision repair activity in extracts of mouse embryonic fibroblasts, DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity, Reconstitution of the DNA base excision‐repair pathway, Generation of single‐nucleotide repair patches following excision of uracil residues from DNA, Repair pathways for processing of 8‐oxoguanine in DNA by mammalian cell extracts, Ionising radiation induces the expression of PARP‐1 and PARP‐2 genes in Arabidopsis, Base damage and single‐strand break repair: mechanisms and functional significance of short‐ and long‐patch repair subpathways, The type of DNA glycosylase determines the base excision repair pathway in mammalian cells, DNA polymerase λ (Pol λ), a novel eukaryotic DNA polymerase with a potential role in meiosis, DNA polymerase λ, a novel DNA repair enzyme in human cells, Evidence for base excision repair of oxidative DNA Damage in chloroplasts of, DNA base excision repair in human malaria parasites is predominantly by a long‐patch pathway, Uracil in DNA – General mutagen, but normal intermediate in acquired immunity, Disparity between DNA base excision repair in yeast and mammals: translational implications, Second pathway for completion of human DNA base excision‐repair: reconstitution with purified proteins and requirement for DNase IV (FEN1), Reconstitution of DNA base excision‐repair with purified human proteins: interaction between DNA polymerase β and the XRCC1 protein, Components of nucleotide excision repair and DNA damage tolerance in, Analysis of class II (hydrolytic) and class I (beta‐lyase) apurinic/apyrimidinic endonucleases with a synthetic DNA substrate, Repair of damaged DNA by Arabidopsis cell extract, Instability and decay of the primary structure of DNA, Post‐translational modification of poly(ADP‐ribose) polymerase induced by DNA strand breaks, Repair of a synthetic abasic site in DNA in a, Repair of a synthetic abasic site involves concerted reactions of DNA synthesis followed by excision and ligation, Synchronous Arabidopsis suspension cultures for analysis of cell‐cycle gene activity, Reconstitution of human base excision repair with purified proteins, Arabidopsis DEMETER‐LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks, Base excision repair of adenine/8‐oxoguanine mispairs by an aphidicolin‐sensitive DNA polymerase in human cell extracts, Aphidicolin‐resistant and ‐sensitive base excision repair in wild‐type and DNA polymerase β‐defective mouse cells, Long patch base excision repair with purified human proteins. Additionally, recent genetic and biochemical evidence has revealed that plant BER performs a key role in epigenetic regulation, through active DNA demethylation initiated by 5‐methylcytosine DNA glycosylases (Agius et al., 2006; Gehring et al., 2006; Morales‐Ruiz et al., 2006). Quantification of the relative fluorescence intensity revealed that fragments corresponding to δ‐elimination products are threefold more abundant than those representing β‐elimination ends (Figure 3a, lanes 5 and 6). Three main observations indicate that repair of a U:G mispair may take place via single‐nucleotide DNA synthesis: (i) dCTP alone is sufficient to support full repair; (ii) only a 29‐nt intermediate is detected in single‐nucleotide reactions; and (iii) the only nucleotide inserted is complementary to the base opposite the U residue. Our results indicate that DNA synthesis after uracil excision is aphidicolin resistant and ddCTP sensitive. In the presence of dCTP as the only deoxynucleotide, a fully repaired product was detected (Figure 4, lane 8), with the concomitant appearance of a 29‐nt fragment on the upper strand, corresponding to the insertion of dCMP in the repair gap (Figure 4, lane 4). Table S1. Identification of a unique insertion in plant organellar DNA polymerases responsible for 5′-dRP lyase and strand-displacement activities: Implications for Base Excision Repair. Homologs of the mammalian flap endonuclease FEN1 are encoded in the genomes of Arabidopsis and Oryza sativa (Kimura and Sakaguchi, 2006), but their role in BER is not known. Uracil repair by Arabidopsis cell extracts is dependent on uracil DNA glycosylase (UDG) activity.DNA duplexes containing either U or THF opposite G were incubated with Arabidopsis cell extracts at 30°C for 3 h in a reaction mixture containing either dCTP or all four dNTPs, both in the absence or presence of 2 U of uracil‐DNA glycosylase inhibitor (Ugi). We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. (a) Schematic diagram of molecules used as DNA substrates. The importance of safeguarding genome integrity in germination and seed longevity. Accidental Dread using: Nether Faerie Dragon (221), Chrominius (212) and Any Pet. The faint bands just above the 28‐nt position (Figure 5, lanes 4 and 12) might represent some β‐elimination product(s) generated by AP lyases. Control AP endonuclease reactions were performed by incubating a DNA containing a natural AP site opposite guanine with human APE1 (10 U) at 37°C for 60 min, supplemented with 2.4 U of human DNA polymerase β when generating a control dRP‐lyase product. It will be important to determine whether these two pathways occur in competition in plant cells, and to identify the factors determining their relative significance in vivo. Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1. Thus, repair of hypoxanthine or ethenoadenine initiated by the ANPG glycosylase is completed via both SP and LP BER (Fortini et al., 1999), whereas excision of 8‐oxoG by OGG1 is preferentially followed by an SP BER (Dianov et al., 1998; Fortini et al., 1999). In this case, the reaction intermediates detected included not only 29‐, but also 30‐ and 31‐nt 5′‐labeled fragments. This strategy was created pre-Shadowlands and has not been tested or adapted. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short‐patch BER) or several nucleotides (long‐patch BER). In any case, our results are compatible with the hypothesis that AP endonuclease‐dependent and ‐independent BER pathways coexist in plant cells, as reported in mammalian cells (Wiederhold et al., 2004). Where indicated, DNA was resuspended in 5 μl of SuRE/Cut Buffer L containing 5 U of HpaII (Roche) and incubated at 37°C for 1 h. Reactions were then stopped by adding 5 μl of 90% formamide and then heating at 95°C for 5 min. Thus, in Saccharomyces cerevisiae most AP sites are repaired through LP BER (Boiteux and Guillet, 2004), whereas in Schizosaccharomyces pombe genetic and biochemical evidence points to the preeminence of the SP pathway (Alseth et al., 2004). We discuss our findings in the light of what is currently known about plant DNA polymerases. The faint bands just above the 28‐nt position (Figure 5, lanes 4 and 12) might represent some β‐elimination product(s) generated by AP lyases. Sterilized seeds of wild‐type Arabidopsis thaliana plants (ecotype Columbia) were plated on 10‐cm‐diameter Petri dishes containing 25 ml of 0.44% (w/v) MS medium (Sigma‐Aldrich, http://www.sigmaaldrich.com), supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar, pH 5.8. In Schizosaccharomyces pombe, for example, genetic and biochemical evidence points to the activity of DNA glycosylase/lyase Nth1 as being mainly responsible for incision at AP sites, which are then processed by the AP endonuclease Apn2 and repaired though an SP subpathway (Alseth et al., 2004).

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