Maintenance of genome integrity by the SMC5/6 complex during deaminase-mediated mutagenesis

Washington University, Saint Louis MO

Investigators

Abstract

PROJECT SUMMARY Cells must maintain the integrity of their genome during mutagenic damage caused by endogenous or exogenous genotoxins. To do so, cells employ a multitude of genome-protective pathways. Cancer cells have a much higher mutation burden than non-malignant cells, and therefore are more genomically unstable. A common source of mutation in cancer is cytidine deamination caused by the endogenously encoded APOBEC3A enzyme. APOBEC3A normally functions as part of the innate immune system to restrict virus infection and retrotransposition, but when dysregulated can act on the cellular genome. Deamination events cause a spectrum of mutagenic outcomes which elicit DNA repair pathways and genome-protective responses. APOBEC3A deaminase activity is the cause of prevalent mutational patterns found in many human cancers, therefore it is imperative to understand the cellular factors that promote or mitigate mutagenesis. Our preliminary data identify the SMC5/6 (Structural Maintenance of Chromosomes 5/6) complex as essential to prevent genotoxicity from APOBEC3A activity. The SMC5/6 complex is highly conserved and has been studied mostly in budding yeast. From yeast models, SMC5/6 is known to play a role in support of both normal and stressed replication forks. APOBEC3A acts primarily on DNA at replication forks, and our data suggest that deaminase activity causes replication stress that is mitigated by the SMC5/6 complex. However, the mechanism by which SMC5/6 protects replication forks from APOBEC3A-mediated damage is unknown. The goals of this proposal are to define the impact of APOBEC3A activity on replication fork progression and the mechanism by which SMC5/6 protects forks from deaminase activity. Aim 1 will define the effect of APOBEC3A deaminase activity on replication fork progression and genome duplication. We will identify the cellular mechanisms that enable replication forks to tolerate obstacles presented by APOBEC3A activity, and how this influences overall mutational burden. Aim 2 will determine how SMC5/6 prevents replication-associated DNA breaks when APOBEC3A is active. Using proteomics, single-molecule imaging, and separation-of-function mutants, we will dissect the specific activities of SMC5/6 that confer replication fork protection. These studies will capitalize on our novel observation of synthetic lethality between APOBEC3A and loss of SMC5/6 to investigate mechanisms of genome protection by the SMC5/6 complex, which remain enigmatic especially in mammalian cells. The impact of the proposed studies will form a foundation for new targeted treatment options by defining mechanisms of DNA damage and genome protection in cancer.