O V A.1 Nucleotide excision repair mechanism in human cells: Analysis by in vitro assays

Nucleotide excision repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified excision repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on nucleotide excision repair in humans.

Download Full-text

Polynuclear ruthenium organometallic compounds induce DNA damage in human cells identified by the nucleotide excision repair factor XPC

Bioscience Reports ◽

10.1042/bsr20190378 ◽

2019 ◽

Vol 39 (7) ◽

Cited By ~ 1

Author(s):

Olivia G. Fast ◽

Brittany Gentry ◽

Liah Strouth ◽

Madison B. Niece ◽

Floyd A. Beckford ◽

...

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Organometallic Compounds ◽

Chemotherapeutic Agents ◽

Human Cells ◽

Nucleotide Excision ◽

Antibiotic Efficacy ◽

In Cells

Abstract Ruthenium organometallic compounds represent an attractive avenue in developing alternatives to platinum-based chemotherapeutic agents. While evidence has been presented indicating ruthenium-based compounds interact with isolated DNA in vitro, it is unclear what effect these compounds exert in cells. Moreover, the antibiotic efficacy of polynuclear ruthenium organometallic compounds remains uncertain. In the present study, we report that exposure to polynuclear ruthenium organometallic compounds induces recruitment of damaged DNA sensing protein Xeroderma pigmentosum Group C into chromatin-immobilized foci. Additionally, we observed one of the tested polynuclear ruthenium organometallic compounds displayed increased cytotoxicity against human cells deficient in nucleotide excision repair (NER). Taken together, these results suggest that polynuclear ruthenium organometallic compounds induce DNA damage in cells, and that cellular resistance to these compounds may be influenced by the NER DNA repair phenotype of the cells.

Download Full-text

Comparative analyses of two primate species diverged by more than 60 million years show different rates but similar distribution of genome-wide UV repair events

BMC Genomics ◽

10.1186/s12864-021-07898-3 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Umit Akkose ◽

Veysel Ogulcan Kaya ◽

Laura Lindsey-Boltz ◽

Zeynep Karagoz ◽

Adam D. Brown ◽

...

Keyword(s):

Nucleotide Excision Repair ◽

Cell Lines ◽

Human Cell ◽

Excision Repair ◽

Mouse Lemur ◽

Repair Mechanism ◽

Damage Repair ◽

Genome Wide ◽

Repair Efficiency ◽

Nucleotide Excision

Abstract Background Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur, Microcebus murinus, in comparison to human. Results We derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation, and analyzed the repair events genome-wide using the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated, and this correlation is stronger for highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do. Conclusion Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.

Download Full-text

Stable and specific association between the yeast recombination and DNA repair proteins RAD1 and RAD10 in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3041-3049.1992 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3041-3049

Author(s):

L Bardwell ◽

A J Cooper ◽

E C Friedberg

Keyword(s):

Dna Repair ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Specific Interaction ◽

Mitotic Recombination ◽

Nucleotide Excision ◽

Specific Association ◽

Recombination Pathway ◽

Cloned Gene

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are two of at least seven genes which are known to be required for damage-specific recognition and/or damage-specific incision of DNA during nucleotide excision repair. RAD1 and RAD10 are also involved in a specialized mitotic recombination pathway. We have previously reported the purification of the RAD10 protein to homogeneity (L. Bardwell, H. Burtscher, W. A. Weiss, C. M. Nicolet, and E. C. Friedberg, Biochemistry 29:3119-3126, 1990). In the present studies we show that the RAD1 protein, produced by in vitro transcription and translation of the cloned gene, specifically coimmunoprecipitates with the RAD10 protein translated in vitro or purified from yeast. Conversely, in vitro-translated RAD10 protein specifically coimmunoprecipitates with the RAD1 protein. The sites of this stable and specific interaction have been mapped to the C-terminal regions of both polypeptides. This portion of RAD10 protein is evolutionarily conserved. These results are the first biochemical evidence of a specific association between any eukaryotic proteins genetically identified as belonging to a recombination or DNA repair pathway and suggest that the RAD1 and RAD10 proteins act at the same or consecutive biochemical steps in both nucleotide excision repair and mitotic recombination.

Download Full-text