Arabidopsis ZDP DNA 3′‐phosphatase and ARP endonuclease function in 8‐oxoG repair initiated by FPG and OGG1 DNA glycosylases

Abstract

SUMMARYOxidation of guanine in DNA generates 7,8-dihydro-8-oxoguanine (8-oxoG), an ubiquitous lesion with mutagenic properties. 8-oxoG is primarily removed by DNA glycosylases distributed in two families, typified by bacterial Fpg proteins and eukaryotic Ogg1 proteins. Interestingly, plants possess both Fpg and Ogg1 homologs but their relative contributions to 8-oxoG repair remain uncertain. In this work we used Arabidopsis cell-free extracts to monitor 8-oxoG repair in wild-type and mutant plants. We found that both FPG and OGG1 catalyze excision of 8-oxoG in Arabidopsis cell extracts by a DNA glycosylase/lyase mechanism, and generate repair intermediates with blocked 3 0 -termini. An increase in oxidative damage is detected in both nuclear and mitochondrial DNA from double fpg ogg1 mutants, but not in single mutants, which suggests that a single deficiency in one of these DNA glycosylases may be compensated by the other. We also found that the DNA 3 0 -phosphatase ZDP (zinc finger DNA 3 0 -phosphoesterase) and the AP(apurinic/apyirmidinic) endonuclease ARP(apurinic endonuclease redox protein) are required in the 8-oxoG repair pathway to process the 3 0 -blocking ends generated by FPG and OGG1. Furthermore, deficiencies in ZDP and/or ARP decrease germination ability after seed deteriorating conditions. Altogether, our results suggest that Arabidopsis cells use both FPG and OGG1 to repair 8-oxoG in a pathway that requires ZDP and ARP in downstream steps.

Phosphoproteomic analysis reveals plant DNA damage signalling pathways with a functional role for histone H2AX phosphorylation in plant growth under genotoxic stress

“…These results are consistent with important roles for ATM post‐translational signalling networks operating in seeds, supported by the known functions of ATM in germination and maintenance of genome integrity in seeds (Waterworth et al ., ). ATM functions to modulate cell‐cycle activity in response to DNA damage, imposing a delay to germination, whereas H2AX is a repair factor and, like several other DNA repair genes, displays hypersensitivity to seed ageing (Waterworth et al ., , ; Córdoba‐Cañero et al ., ). Phosphoproteome analysis of germinating Lotus japonicas also identified numerous phosphorylated ATM/ATR SQ consensus motifs (Ino et al ., ), further supporting the importance of these post‐translational signalling networks at the seed stage of the plant lifecycle.…”

Oxidative stress and antioxidative systems: recipes for successful data collection and interpretation

“…Double mutants lacking OGG1 and another enzyme involved in 8‐oxo‐G repair showed increased DNA damage, and both single and double mutants also lost germination capacity more quickly than wild‐type seeds during ageing (Cordoba‐Cañero et al . ). Guanine bases in RNA can also undergo this modification.…”

“…A small increase in 8-oxo-G was also reported in leaves of catalase-deficient plants grown in oxidative stress conditions, but no increase was observed in a marker for DNA double strand breaks that can accompany this modification (Vanderauwera et al 2011). Accurate estimation of products of oxidative modification of DNA is not trivial, and high basal levels of 8-oxo-G have been reported in plants as in other organisms, possibly because of DNA oxidation during extraction (Dizdaroglu et al 2002;Bazin et al 2011;Vanderauwera et al 2011;Cordoba-Cañero et al 2014).…”

“…Double mutants lacking OGG1 and another AO, ascorbate oxidase; ADH, alcohol dehydrogenase; ASC, ascorbate (reduced form); DCPIP, dichlorophenolindophenol; DHA, dehydroascorbate; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol; EtOH, ethanol; GSH, glutathione (reduced form); GR, glutathione reductase; GSSG, glutathione disulphide; 6-PG, 6-phosphoglucono-γ-lactone; G6P(DH), glucose-6-phosphate (dehydrogenase); PMS, phenazine methosulphate; VPD, 2-vinylpyridine. More details are given on methods in Boxes 8-10. enzyme involved in 8-oxo-G repair showed increased DNA damage, and both single and double mutants also lost germination capacity more quickly than wild-type seeds during ageing (Cordoba-Cañero et al 2014). Guanine bases in RNA can also undergo this modification.…”

MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

“…2). All mutant lines were originally 452 generated in a Col-0 background with exception of the fpg and ogg1 mutants, which were in a 453 Landsberg erecta (Ler) background (11,60). F2 plants were screened with allele-specific PCR 454 markers (Table S6) to identify individuals that were homozygous for the mutant allele and others that 455 were homozygous for the wild type allele.…”