Identification of proteins involved in nucleotide excision repair in arabidopsis thaliana

Plant genomes are constantly challenged by environmental genotoxins and ultraviolet (UV) radiation, requiring efficient DNA repair mechanisms. Nucleotide excision repair (NER) removes bulky DNA lesions, such as cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine photoproducts (6-4pp) that are caused by UV radiation. NER functions by two sub-pathways: global genome repair (GG-NER) and Transcription-coupled repair (TC-NER). GGNER scans the entire genome to identify lesions while TCNER removes lesions from actively transcribed strands. In mammalian NER, CSB is responsible for identifying the stalled RNA polymerase II at the lesion site and recruiting downstream NER proteins. In Arabidopsis thaliana Chromatin remodelling 24 (Chr24) and Chromatin remodelling 8 (Chr8) are homologs of CSB, but their role in NER remained poorly understood. We utilized Excision repair sequencing (XR-seq) methodology to generate genome-wide repair maps in Chr8 mutants and Wild-type (WT). Chr8 mutants showed loss of repair across gene bodies and no template-strand bias. In contrast, WT exhibited strong repair signals on transcribed strands across gene bodies. These results confirm the role of Chr8 in plant TC-NER and highlight its evolutionary conservation. Understanding the NER mechanism in plants is essential for developing UV-tolerant genotypes and answering how plants maintain their genome integrity under stressed environments. In eukaryotes, DNA is packed around histone proteins, forming a compact chromatin structure creating a higher level of genome organization. This compact chromatin structure act as a natural barrier restricting NER machinery from accessing DNA damage across those regions. ATP-dependent chromatin remodelling complexes (CRCs) are responsible for repositioning and restructuring of nucleosomes, providing the access to repair machinery to identify and repair DNA damage. In plants, relationship between NER and CRCs is poorly understood. So, we investigated the relationship of four chromatin modellers in Arabidopsis thaliana: Chromatin remodelling 11 (Chr11), Chromatin remodelling 12 (Chr12), Chromatin remodelling 17 (Chr17) and Chromatin remodelling 23 (Chr23). By utilizing the XR-seq method, we generated XR-seq libraries for CRC mutants in Arabidopsis and compared the repair profiles across nine chromatin states, including open chromatin and repressive chromatin states. CRC mutants exhibited reduced repair in repressive chromatin states especially Heterochromatin AT/GC rich regions and polycomb chromatin. Surprisingly, all CRC mutants exhibited wild-type phenotype across gene bodies and open chromatin states. These results indicate that CRCs facilitate the NER in heterochromatin regions and are an integral part of NER machinery.