Abstract 3012: The Snm1B/Apollo DNA nuclease functions in resolution of replication stress and maintenance of genome stability

Abstract

Abstract A critical aspect of normal cellular processes is the maintenance of genomic stability. Defects in cellular responses to DNA damage can lead to an accumulation of unrepaired or misrepaired lesions and ultimately, increased genome instability. Genomic DNA is constantly damaged through exposure to exogenous agents as well as during endogenous processes, including DNA replication. Progression of the replication fork can be impaired by structural or physical blocks leading to fork stalling. If stalled forks are not properly restarted or repaired, they can collapse, leading to chromosomal damage including breaks, deletions, and translocations. Therefore, replication stress associated DNA damage has been hypothesized as one important source of genome instability associated with cancer initiation and progression. Cells have evolved intricate mechanisms to detect, signal, and resolve stalled replication forks; however, the molecular events involved in these fundamental processes have not been fully defined. My studies are focused on the DNA nuclease SNM1B/Apollo which plays critical roles in repairing stalled or blocked replication forks and also in telomere maintenance. We previously reported that SNM1B functions within the Fanconi anemia (FA) pathway, which is required for efficient stabilization of stalled replication forks. Mutations in the 16 known FA genes cause Fanconi anemia, a human genome instability disorder characterized by cancer predisposition, progressive bone marrow failure, and developmental defects. It is emerging that the SNM1B locus is genomically altered or mutated in a variety of cancer types and has been mapped at a chromosomal breakpoint associated with Wilms tumor. Thus, SNM1B likely plays important roles in the prevention of potential oncogenic chromosomal anomalies; however, its precise functions during DNA repair processes are not well understood. My current research is focused on elucidating the importance of the nuclease activity of SNM1B in stabilizing and repairing stalled replication forks and how these functions are regulated. Interestingly, SNM1B possesses the monoubiquitination consensus sequence, RKQL, found in the key FA protein, FANCD2. I found that substituting arginine for lysine within the consensus sequence results in defective localization of the mutant SNM1B protein to stalled forks as well as impaired FANCD2 recruitment to sites of damage, indicating that this residue has functional importance in the repair of stalled forks. Using a siRNA knockdown approach in human cell lines, I determined that SNM1B depletion significantly increases fork stalling and single-stranded DNA regions, even in unperturbed cells. Altogether, my recent studies led us to propose that functions of the SNM1B nuclease during the repair of stalled replication forks ensures successful replication of the genome and thus, prevents potentially oncogenic chromosomal aberrations. Citation Format: Ishita Das, Jennifer Mason, JoAnn Sekiguchi. The Snm1B/Apollo DNA nuclease functions in resolution of replication stress and maintenance of genome stability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3012. doi:10.1158/1538-7445.AM2015-3012