Dna2 Responds to Endogenous and Exogenous Replication Stress in Drosophila melanogaster

<b>Background/Objectives</b>: DNA2 is a conserved nuclease-helicase that plays a crucial role in DNA replication and repair by responding to replication stress. Previous studies have established the role of DNA2 in Okazaki fragment processing, the recovery of stalled replication forks, and double-strand break repair. This study investigates the role of <i>Drosophila melanogaster</i> Dna2 in response to exogenous DNA damage and replication stress as well as during developmental stages involving intensive DNA replication. <b>Methods</b>: We used the <i>Drosophila</i> mutant alleles, <i>Dna2^D1</i> and <i>Dna2^D2</i>, which differ in the presence of the helicase 1A domain, to assess sensitivity to mutagens that cause various types of replication stress and DNA damage. We examined reproductive fitness through Mendelian ratio calculations, fecundity, egg viability assays, and assessed DNA damage via immunostaining of ovarian germaria. Lifespan assays were also conducted to examine adult survival. <b>Results</b>: <i>Dna2</i> mutants demonstrated significant sensitivity to replication stress induced by MMS, hydroxyurea, topotecan, and nitrogen mustard. <i>Dna2^lS/S1</i> mutants exhibited higher survival than <i>Dna2^lS/D2</i> upon exposure to topotecan and bleomycin, suggesting a possible helicase-specific role in damage response. Mutants exhibited decreased fecundity, reduced egg viability, and elevated DNA damage in mitotically active germline cells. Adult lifespan was not reduced in <i>Dna2</i> mutants, implying potential compensatory stress-response mechanisms. <b>Conclusions</b>: Our findings support a requirement of Dna2 in managing replication stress during critical developmental phases in <i>Drosophila</i>. These insights clarify the nuanced contributions of the nuclease and helicase domains of DNA2, suggesting potential domain-specific functions in genomic stability and repair mechanisms. This work provides a foundation that will enable future researchers to further dissect the complex roles of DNA2 in replication and repair pathways.