Repair of protein-linked DNA double strand breaks: Using the adenovirus genome as a model substrate in cell-based assays
| Autor: | Matthew D. Weitzman, Nicole I. Orazio, Tony Hunter, Brandon J. Lamarche, Zhongsheng You, Brittany Goben, Jill Meisenhelder |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
Spo11
DNA Repair Concatemer Genome Viral Biochemistry Article Adenoviridae 03 medical and health sciences chemistry.chemical_compound Endonuclease 0302 clinical medicine Humans DNA Breaks Double-Stranded Molecular Biology 030304 developmental biology 0303 health sciences MRE11 Homologue Protein Endodeoxyribonucleases biology Oligonucleotide Adenovirus genome BRCA1 Protein fungi Nuclear Proteins Proteins Cell Biology Cell biology Non-homologous end joining HEK293 Cells chemistry 030220 oncology & carcinogenesis Gene Knockdown Techniques biology.protein Homologous recombination Carrier Proteins DNA |
| Zdroj: | DNA Repair (Amst) |
| Popis: | The DNA double strand breaks (DSBs) created during meiotic recombination and during some types of chemotherapy contain protein covalently attached to their 5' termini. Removal of the end-blocking protein is a prerequisite to DSB processing by non-homologous end-joining or homologous recombination. One mechanism for removing the protein involves CtIP-stimulated Mre11-catalyzed nicking of the protein-linked strand distal to the DSB terminus, releasing the end-blocking protein while it remains covalently attached to an oligonucleotide. Much of what is known about this repair process has recently been deciphered through in vitro reconstitution studies. We present here a novel model system based on adenovirus (Ad), which contains the Ad terminal protein covalently linked to the 5' terminus of its dsDNA genome, for studying the repair of 5' protein-linked DSBs in vivo. It was previously shown that the genome of Ad mutants that lack early region 4 (E4) can be joined into concatemers in vivo, suggesting that the Ad terminal protein had been removed from the genome termini prior to ligation. Here we show that during infection with the E4-deleted Ad mutant dl1004, the Ad terminal protein is removed in a manner that recapitulates removal of end-blocking proteins from cellular DSBs. In addition to displaying a dependence on CtIP, and Mre11 acting as the endonuclease, the protein-linked oligonucleotides that are released from the viral genome are similar in size to the oligos that remain attached to Spo11 and Top2 after they are removed from the 5' termini of DSBs during meiotic recombination and etoposide chemotherapy, respectively. The single nucleotide resolution that is possible with this assay, combined with the single sequence context in which the lesion is presented, make it a useful tool for further refining our mechanistic understanding of how blocking proteins are removed from the 5' termini of DSBs. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|