Seth Wallerstein

In response to the constant formation of lesions that compromise the integrity of their DNA, cells employ numerous mechanisms designed to resolve these lesions. When these mechanisms fail, genomic integrity can break down and generate genetic abnormalities such as mutations, aneuploidy, incompletely replicated chromosomes, and accumulation of double-strand breaks (DSBs). One important factor in the pathway of DNA repair is the DNA2 nuclease/helicase. In yeast, this enzyme plays a role in a wide range of processes, including Okazaki fragment maturation, processing of stalled replication forks, and end resection at DNA double-strand breaks and in generation of the telomeric G-overhang. However, the precise mechanisms of action and essential function of DNA2 in human cells remain unknown, largely a result of biological differences between yeast and humans, especially in the context of telomere maintenance. Here, I describe the generation of DNA2 conditional knockout cell lines that can be used to investigate the impact of the absence of DNA2. These conditional knockout cell lines were established by incorporation of loxP sites flanking exon 7 of DNA2 and random integration of the CreERT2 fusion gene. Cells carrying these modifications become sensitive to 4-OHT due to recombination that irreversibly removes DNA2 exon 7 from the genome. We anticipate that following confirmation of the functionality of these cell lines, most notably the loss of DNA2 protein in addition to the loss of genomic DNA upon 4-OHT treatment, we will use them as tools to investigate the DNA2-null phenotype and to define the essential function of DNA2 in ways which were not previously possible. Specifically, we will analyze the role of DNA2 in telomere maintenance and other aspects of the maintenance of genome integrity. We also envision using the cell lines in the future to carry out screens to evaluate the interaction of DNA2 with other repair proteins.