Single‐nucleotide and long‐patch base excision repair of DNA damage in plants

Abstract

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell-free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′- or 3′-blocked ends, respectively. 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). 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.

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

“…6B). Consistent with previously reported observations (25), wild-type Arabidopsis extracts are capable of performing full BER of the DNA containing U:G or THF:G, in the presence of either dCTP or of all four dNTPs (Fig. 6B).…”

“…5A). We have previously reported the processing of AP sites by Arabidopsis AP lyases in the absence of Mg 2ϩ (25). No UDG activity was detected in the extract when the repair reaction was performed in the presence of the Family-1 enzyme inhibitor Ugi (Fig.…”

“…6A). Repair is detected as conversion of the HpaII-resistant DNA substrate to a form susceptible to digestion, and fully repaired products are visualized by the emergence of a 21-nt labeled fragment following polyacrylamide gel electrophoresis (25).…”

“…We have recently developed an in vitro assay to monitor BER of damaged bases in Arabidopsis (25). In this assay, plant cell extracts are incubated with a DNA duplex that contains a single base modification within an HpaII recognition site on the upper strand and is labeled at the 5Ј-end of the lower strand (Fig.…”

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).…”