Using Arabidopsis Cell Extracts to Monitor Repair of DNA Base Damage In Vitro

Abstract

Base excision repair (BER) is a major pathway for the removal of endogenous and exogenous DNA damage. This repair mechanism is initiated by DNA glycosylases that excise the altered base, and continues through alternative routes that culminate in DNA resynthesis and ligation. In contrast to the information available for microbes and animals, our knowledge about this important DNA repair pathway in plants is very limited, partially due to a lack of biochemical approaches. Here we describe an in vitro assay to monitor BER in cell-free extracts from the model plant Arabidopsis thaliana. The assay uses labeled DNA substrates containing a single damaged base within a restriction site, and allows detection of fully repaired molecules as well as DNA repair intermediates. The method is easily applied to measure the repair activity of purified proteins and can be successfully used in combination with the extensive array of biological resources available for Arabidopsis.

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

“…The 3′‐P fragment disappeared when reactions were performed in the presence of Mg 2+ (Figure b, lanes 9 and 13), but a significant proportion was accumulated in control reactions catalyzed by extracts from zdp mutants (Figure b, lanes 7 and 14), which are deficient in the major DNA 3′‐phosphatase activity detected in Arabidopsis (Martínez‐Macías et al ., ). Additional, shorter fragments were observed in the presence of Mg 2+ , as a result of exonucleolytic degradation in the absence of dNTPs (Córdoba‐Cañero et al ., ). Extracts from ogg1 mutants generated products similar to those of WT plants (Figure b, lane 4).…”

“…Uracil and AP site BER assays were performed as previously described (Córdoba‐Cañero et al ., , ). DNA ligation assay of duplexes containing a nick in the 5′ alexa fluor‐labeled upper strand were performed as uracil and AP site BER assays, but in the absence of dNTPs.…”

“…8‐oxoG DNA substrates contained a single 8‐oxoG residue within a Hae III restriction site, with the purpose of detecting full repair as conversion to a form susceptible to digestion, as previously described for U and AP sites within an Hpa II target (Córdoba‐Cañero et al ., , ). However, we found that Hae III digestion was incompletely blocked by 8‐oxoG (Figure S4).…”

The DNA Repair Protein XRCC1 Functions in the Plant DNA Demethylation Pathway by Stimulating Cytosine Methylation (5-meC) Excision, Gap Tailoring, and DNA Ligation*

“…The Arabidopsis mutant line SALK_027362 harboring a T-DNA insertion in the XRCC1 gene (30) was a kind gift from Charles White (Blaise Pascal University, France). Cells extracts were prepared from snap-frozen 15-day-old seedlings as described previously (31,32).…”

Preparation of Efficient Excision Repair Competent Cell-Free Extracts from C. reinhardtii Cells

“…During ER, the damaged DNA is recognized, excised as a patch of single strand encompassing not only the lesion but also a few normal nucleotides on either side of it, and replaced with new nucleotides via repair DNA synthesis [7] , [8] . Therefore, most in vitro ER reactions have been reliably assayed by the accompanying incorporation of labeled dNTP precursors specifically in the damaged DNA [9] – [11] .…”