Translesion Synthesis DNA Polymerases in Mitigating Replication Stress

About This Grant

Abstract Hematopoietic stem cells (HSCs) require means to maintain genome stability to sustain a lifelong supply of blood cells. This is prominently illustrated by Fanconi Anemia (FA), which results in bone marrow failure (BMF) and increased leukemia incidence. We showed that Lnk deficiency increases HSCs in FA mutant mice and primary FA patient cells. A critical question is how LNK controls DNA replication-associated DNA damage. Mice lacking a central component of the FA pathway, Fancd2, show severely compromised HSC activity in bone marrow transplant (BMT) in vivo. Fancd2-/- HSCs and progenitors (HSPCs) cannot sustain ex vivo culture. This HSPC attrition is due to heightened DNA damage and chromosome abnormalities, causing cessation of cell proliferation and increased apoptosis. We showed that Lnk deficiency reduces DNA damage associated with replication stress in HSPCs, particularly in the FA background. Lnk deficiency suppresses single-strand DNA gaps, thus reducing ATR and p53 checkpoint activation that is linked to HSC attrition. One of key mechanisms of DNA damage tolerance in cells is translesion synthesis (TLS). TLS polymerases (Polh, Poli, Polk, and REV1) are specialized DNA polymerases that facilitate replication in the presence of DNA lesions or natural replication fork barriers that stall the replicative polymerases. Their unique ability to replicate over difficult-to-replicate regions makes them critical for maintaining genome stability. However, the complex regulation of DNA replication and repair in hematopoietic cells is not well-established. We discovered that the enhanced HSC fitness from Lnk deficiency is associated with increased TLS activity via REV1, a scaffold protein that brings multiple TLS polymerases to stressed replication forks. We thus hypothesize that Lnk deficiency promotes HSC expansion by enhancing the activity of TLS DNA polymerases to mitigate replication stress. We aim to investigate mechanisms underlying LNK regulation of TLS polymerases and their functional significance to HSC fitness in the context of FA. In Aim 1, we will investigate the role of TLS polymerases in regulating HSC function and replication stress using mice deficient for Rev1 and various TLS polymerases. In Aim 2, we will determine how LNK regulates TLS polymerases and replication factors upon replication stress in HSPCs. We will examine whether LNK impacts TLS-mediated DNA replication using TLS reporters ex vivo and in vivo. We will decipher how Lnk deficiency impacts replication fork dynamics using a combination of complementary genetics, proteomics, and pharmacological approaches. In Aim 3, we will determine the role of TLS in replication stress relief in human FA HSPCs. We will investigate whether TLS inhibition exacerbates the growth defects of human FA HSPCs and, conversely, whether overexpression of components of the TLS pathway promotes the growth of human HSPCs from healthy donors and FA patients. Our work will deepen understanding of mechanisms underpinning stem cell attrition in healthy and disease settings and may unveil new therapeutic strategies to improve HSC fitness for the treatment of FA and for stem cell-based therapy in general.