Repair of Damaged DNA by Arabidopsis Cell Extract

Abstract

All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light-and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue-induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay. INTRODUCTIONThe genomes of all living organisms are constantly subjected to a wide range of genotoxic stresses induced by environmental factors (e.g., UV-B irradiation, bacterial and fungal toxins) as well as by the intermediate products of normal cellular metabolism (e.g., alkylating and oxidizing agents). These can lead to the formation of different types of DNA damage, the persistence of which can block DNA replication and transcription or cause cell cycle arrest and apoptosis (Britt, 1999;Lindahl and Wood, 1999). Incorrect repair can result in heritable point mutations or gross rearrangements such as deletions and insertions. To keep the integrity of their genomes, all organisms have evolved protective mechanisms of repair of a broad variety of DNA lesions. According to the mode of action, the substrate specificity, and the size of the excised DNA fragment, these pathways generally have been classified as direct repair, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (reviewed by Friedberg, 1996;Sancar, 1996;Wood, 1996;Lindahl and Wood, 1999). These mechanisms were first described in bacteria and later characterized extensively in yeast and mammals (Sancar, 1996;Laat et al., 1999;Le Page et al., 2000;Memisoglu and Samson, 2000). Unfortunately, with the exception of light-dependent reversion of UV light-induced pyrimidine dimers by photolyases, very little is known about DNA repair pathways in plants (reviewed by Vonarx et al., 1998; Britt, 1999).To study different types of DNA repair mechanisms in vitro, the formation of pathway-specific DNA lesions is required. For the study of NER, we chose the following DNAdamaging agents: UV-C and cis -diamminedichloroplatinum II (cisplatin). Additionally, methylene blue-treated DNA was used to distinguish the relative contributions of ...

CENTRIN2 Modulates Homologous Recombination and Nucleotide Excision Repair in Arabidopsis[W]

“…Surprisingly the AtRAD1 gene, encoding an endonuclease, which together with Rad10p (ERCC1) is involved in the 59 incision of the UV damage, was found to be upregulated in the Atcen2 plants compared with the control. These results are in apparent conflict with the work of Fidantsef et al (2000) and Li et al (2002) who have shown that RAD1-deficient Arabidopsis plants exhibited higher UV-C sensitivity and reduced efficiency of dark repair of UV-induced DNA damage. Therefore, our data suggest that the increased load of damage, because of downregulation of photolyase and to lower NER efficency, may be responsible for the increased Rad1 mRNA level.…”

“…To test this directly, cell extracts of P2.4, RNAi, and IC9 plants were used in the in vitro DNA repair assay described by Li et al (2002). This assay measures the efficiency of incorporation of DIG dUTP in a UV-C damaged plasmid in the presence of plant cell extracts.…”

“…The pGEX and pBSK plasmids were linearized and purified as described by Li et al (2002). The pGEX-linearized plasmid was UV-C damaged (450 J/m 2 ) using the Stratalinker (Stratagene, La Jolla, CA).…”

“…The in vitro repair assay according the method described by Li et al (2002) was performed on cell extracts from the homozygous P2.4 mutant, RNAi plants, overexpressing plants, and from both the IC9 line and wildtype plants as control. The pGEX and pBSK plasmids were linearized and purified as described by Li et al (2002).…”

Roles of Arabidopsis AtREV1 and AtREV7 in Translesion Synthesis

“…Simpson and Sale (2003) suggested that Rev1 and TLS are critical for efficient tolerance or repair of ICLs and that it is likely involved in other pathways. In higher plants, many genes involved in NER and recombinant repair have been isolated, and some of them were found to be involved in tolerance to DNA cross-linkers (Xu et al, 1998;Li et al, 2002). Therefore, it is possible that TLS works synergistically with NER and/or recombinant repair in plants.…”