Mechanistic Analysis of the Oligomerization States of DmGen in vitro Using Atomic Force Microscopy (2023)

Undergraduate: Juhi Salunke

Faculty Advisor: Dorothy Erie
Department: Chemistry

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DNA damage can happen due to a variety of environmental agents, such as oxidative and UV damage that can lead to DNA mutations within the genome which is linked to disease etiologies. DNA damage repair is an important pathway that helps modulate these damages that occur in DNA replication. One such repair pathway acts in homologous recombination, which acts to resolve DNA intermediates that result from repair of DNA damage in double strand breaks and interstrand crosslinking. A variety of enzymes function along this repair pathway to resolve these DNA intermediates, one such protein being Drosophila Melanogaster Gen (DmGen), an endonuclease protein. In past studies, this endonuclease has been shown to mirror the human homolog GEN1 endonuclease in DNA damage repair. In this study, we will be investigating the mechanism of DmGen by observing how it binds to flapped DNA substrates, which act as a double stranded break DNA intermediate. These findings will help to elucidate the oligomerization and bending of substrates within in vitro studies. By using AFM, we imaged and analyzed how DmGen binds to 5’ flap DNA substrate to better understand the overlying mechanism in which DmGen resolves flapped DNA intermediates. This analysis revealed a distribution of Gen between monomer and dimer states with possible differences in binding angle. These findings from this eukaryotic system centered on DmGen will function to deepen our understanding of human GEN1 and potentially other eukaryotic resolvases in the context of DNA damage repair.

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