Monitoring base excision repair in Chlamydomonas reinhardtii cell extracts

• Published in: DNA repair Vol. 65; pp. 34 - 41
• Main Authors: Morales-Ruiz, Teresa, Romero-Valenzuela, Álvaro C., Vázquez‐Grande, Vanessa M., Roldán-Arjona, Teresa, Ariza, Rafael R., Córdoba‐Cañero, Dolores
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-05-2018
• Subjects: Base excision repair; Chlamydomonas; Chlamydomonas reinhardtii - genetics; Chlamydomonas reinhardtii - metabolism; DNA Damage; DNA Repair; DNA, Plant - metabolism; Uracil - metabolism; DTT; Ugi; Base excision repair; DNA repair; BER; Chlamydomonas; AP; UNG
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2018.02.011

•Base excision repair can be detected in cells extracts of Chlamydomonas reinhardtii.•An uracil DNA glycosylase belonging to Family-1 initiates BER of uracil.•Abasic sites may be processed both by AP lyases and AP endonucleases.•Full repair occurs by both single-nucleotide insertion and long-patch DNA synthesis.
Base excision repair (BER) is a major defense pathway against spontaneous DNA damage. This multistep process is initiated by DNA glycosylases that recognise and excise the damaged base, and proceeds by the concerted action of additional proteins that perform incision of the abasic site, gap filling and ligation. BER has been extensively studied in bacteria, yeasts and animals. Although knowledge of this pathway in land plants is increasing, there are no reports detecting BER in algae. We describe here an experimental in vitro system allowing the specific analysis of BER in the model alga Chlamydomonas reinhardtii. We show that C. reinhardtii cell-free extracts contain the enzymatic machinery required to perform BER of ubiquitous DNA lesions, such as uracil and abasic sites. Our results also reveal that repair can occur by both single-nucleotide insertion and long-patch DNA synthesis. The experimental system described here should prove useful in the biochemical and genetic dissection of BER in algae, and may contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways in photosynthetic eukaryotes.