Nucleotide Excision Repair Factor-XPG

Husna, Amity University Kolkata

Nucleotide Excision Repair (NER) is a repair pathway of the genomic DNA. The genomic DNA can be damaged due to a variety of endogenous and environmental mutagens such as exposure of the skin cells to UV radiations from the sun. Such DNA damage can be repaired by the nucleotide excision repair mechanism.

Any mutation of the NER pathway can lead to various inherited diseases such as Xeroderma pigmentosum (XP). The NER pathway on the mammalian cells involves various major proteins out of which, one is Factor XPG- a protein XPG is named after xeroderma pigmentosum.

XPG is an Endonuclease protein that belongs to the flap endonuclease family of enzymes that can cleave DNA at specific structural sites (1,2). For example, DNA lesions induced by UV radiations result in the distortion of the double helix, so during the nucleotide excision repair (NER) process, it is cleaved by the XPG at 3’ end and by XPF at 5’end.  XPG not only participates in the nucleotide excision repair process, but also in basal transcription, and in the processing of RNA/DNA hybrids (R-loops). 

The mechanism which regulates the association of chromatin to XPG during the process of basal transcription previously unknown. So, the chromatin association to the XPG was investigated in a study both during basal transcription or during the normal cell growth and after the transcriptional stress or inhibition of the transcription.

The analysis was performed in the following ways :-

Chromatin Association of XPG:  In order to analyse the XPG fraction which was chromatin-bound in the absence of DNA damage, an in-situ lysis procedure was applied to detect the protein which are chromatin-associated by immunofluorescence. Immunofluorescence was performed in the proliferating HaCaT keratinocytes and in LF-1 fibroblast sites.

Acetylation of the chromatin-bound XPG: Since some modifications were present even in the absence of DNA damage in the LF-1 cells, they were observed in the absence of UV irradiation too. So, the acetylation of the XPG protein and its interaction with p300/CBP in nuclear extracts of proliferating HaCaT cells was analyzed. Immunoprecipitation Assays were performed as well, in which an antibody to CBP was taken. 

In order to investigate if the general inhibition of transcription would affect XPG acetylation, LF-1 cells were treated with DRB, which is an inhibitor of RNA pol II, and after the extraction of protein, an immunoprecipitation (Ip) was performed with the anti-acK antibody.

Inhibition of Transcription or Transcriptional stress can Induce XPG Accumulation and relocalization:

LF-1 cells were treated with the RNA polymerase II (pol II) inhibitors such as AD or DRB, or with the topo I inhibitor CPT, in addition to C646. When determined by Western Blot Analysis, all of these compounds induced a significant increase in the amount of chromatin-bound XPG, as compared to untreated control cells. 

In addition, the loss of p300 and CBP KAT activity mediated by RNA interference or by chemical inhibitor C646 [29] also resulted in XPG accumulation and relocalization, similar to transcription inhibitors.

XPG Relocation Occurs in combination with Histone γ-H2AX and R-Loop Formation:

The Chromatin accumulation and clustered redistribution occurred in concomitance i.e., in combination with R-loop formation and histone γ-H2AX appearance. Analysis with PLA (Proximity Ligation Assay) showed that XPG was in close proximity to R-loops, which suggests that relocation induced by transcriptional stress leads to the recruitment of XPG in the R-loop sites. 

The results show that the relocation of XPG to nuclear clusters is caused by transcriptional stress. This suggests that the new distribution reflects the processing of R-loops.

Also read:Takotsubo cardiomyopathy – “Breaking hearts” linked to Covid

Source: Scalera C, Ticli G, Dutto I, Cazzalini O, Stivala LA, Prosperi E. Transcriptional Stress Induces Chromatin Relocation of the Nucleotide Excision Repair Factor XPG. International Journal of Molecular Sciences. 2021; 22(12):6589. https://doi.org/10.3390/ijms22126589

• The Corrosion Prediction from the Corrosion Product Performance
• Nitrogen Resilience in Waterlogged Soybean plants
• Cell Senescence in Type II Diabetes: Therapeutic Potential
• Transgene-Free Canker-Resistant Citrus sinensis with Cas12/RNP
• AI Literacy in Early Childhood Education: Challenges and Opportunities