Title : Identification of Arabidopsis proteins involved in nucleotide excision repair
Abstract:
The intricate DNA repair mechanisms organisms employ to rectify damage induced by environmental factors, such as UV rays, play a pivotal role in maintaining genomic stability. UV-induced damage leads to bulky lesions, like CPD and 6-4PP, followed by the subsequent enrichment of specific nucleotides (TT/CT/CC) at the damage site. Global Genome Repair (GG-NER) and Transcription-Coupled Nucleotide Excision Repair (Tc-NER) are two distinct pathways that facilitate DNA repair in nature. In mammals, Tc-NER involves the recruitment of CSA and CSB repair proteins when RNA polymerase halts at the damage site during transcription, resulting in transcription arrest and a repair bubble. This repair bubble facilitates various repair processes, including endonucleolytic incisions, DNA synthesis, ligation, histone modification, and the resumption of transcription. This study aims to identify the proteins engaged in transcription-coupled DNA repair in Arabidopsis, offering insights into repair dynamics using the XR-seq method. Key proteins in Tc-NER for damage recognition are CSA, CSB, and XPA, while XPF and XPG serve as endonucleases responsible for DNA excision. Arabidopsis harbors homologs of key repair proteins like CSA and CSB (CSA1, CSA2, Chr24, and Chr8) for damage recognition, as well as XPF and XPG endonucleases (UVH3, UVH1, and ERCC1). The absence of XPA in Arabidopsis suggests the presence of alternate proteins governing XPA's pivotal role in nucleotide excision repair and transcription-coupled repair in plants. Comparative protein alignments of these homologs underscore potential functional differences. Utilizing the XR-seq methodology allows the generation of a genome-wide DNA repair map in NER knockout mutant genotypes. The main steps of XR-seq involve isolating excised oligomers using methods such as cell lysis, fractionation, immunoprecipitation with damage-specific antibodies, DNA damage reversal via photoreactivation, PCR amplification, and sequencing library preparation. This research contributes to a comprehensive understanding of plant DNA repair processes, shedding light on the unique mechanisms employed by Arabidopsis to maintain genomic integrity amidst DNA damage.
