The genome is constantly exposed to genotoxic damage from endogenous and exogenous sources. Sources of exogenous DNA damage range from natural genotoxins?of which UV light is the primordial example?to products of human activity, such as industrial genotoxic chemicals that are permitted to accumulate in the environment. Damage from such genotoxins can lead to direct chemical alteration of DNA bases (base damage) or chemical modifications of the DNA sugar phosphate backbone (DNA adduction). High volume, efficient DNA repair processes such as base excision repair (BER) and nucleotide excision repair (NER) evolved to repair these types of DNA lesions. Although largely error-free pathways, both BER and NER generate potentially hazardous DNA intermediates, in the form of DNA nicks or single stranded (ss)DNA gaps. If encountered during S phase, collision of a replication fork with the unligated nick converts it to a one-ended double strand break. This presents a significant challenge to the stability of the genome, since DSBs are innately dangerous lesions and one-ended DSBs in particular lack a partner for ligation. The repair mechanisms induced by replication fork collapse at a DNA nick are increasingly well understood in model organisms and, in yeast, are known to entail engagement of ?break-induced replication? (BIR)?an error-prone replicative response initiated at one-ended DSBs. Despite the importance of repair of collapsed replication forks, this process is very poorly understood in mammalian cells, due to the absence of tractable tools. In this proposal, we will develop two new tools aimed at triggering a DNA nick at a defined chromosomal locus in mammalian cells. We will quantify chromosome breakage at the collapsed fork and will measure homologous recombination (HR), including BIR-like ?long tract? gene conversions, triggered by fork collapse at the DNA nick. By using HR reporters that the Scully lab developed for simultaneously quantifying conventional short tract HR and aberrant long tract HR, we will begin to investigate the extent to which fork collapse skews HR in favor of BIR-like responses in mammalian cells. Success in this proposal will establish valuable tools for understanding how environmental genotoxins promote genomic instability in mammalian cells.
Every time a cell divides, it must duplicate the DNA that contains the blueprint of the cell. If this process is interrupted, the cell must bring to bear an exquisitely tuned machinery of DNA repair and other activities to prevent DNA damage, mutation and cancer. In work described here, we will develop powerful new tools to study how damaged replicating DNA is repaired, insights from which will help to reveal how environmental genotoxins lead to mutation and human disease.
| Willis, Nicholas A; Frock, Richard L; Menghi, Francesca et al. (2017) Mechanism of tandem duplication formation in BRCA1-mutant cells. Nature 551:590-595 |
