DNA Damage Response and Replication Stress in Mouse Embryonic Stem Cells

Abstract

DNA replication is a central cellular process that allows duplication of the genetic material before its proper segregation during cell division. The DNA damage response (DDR) protects cells from deleterious mutations during replication and helps maintain genome stability in face of exogenous genotoxic stress. Such pathways must be particularly robust in stem cells, since they are constantly self-renewing and capable of differentiating into all other specialized cells. The main function of embryonic stem cells (ESCs) is to proliferate and differentiate into multiple cell types spatiotemporally, without compromising on their self-renewal capacity. The high proliferative capacity of ESCs is often coupled to rapid G1-S transition and elevated levels of CDKs and other cell cycle regulators. In this study, we show that mouse ESCs surprisingly experience endogenous DNA replication stress (RS), which is characterized by high basal levels of the ATR-dependent DDR marker γH2AX, chromatin recruitment of the single stranded DNA (ssDNA) binding proteins RPA and Rad51, accumulation of ssDNA gaps/nicks, increased replication fork reversal and slow fork progression. Strikingly, all these hallmarks of RS are quickly lost upon induction of differentiation, before cells stop proliferating. Furthermore, PARP1 activity - previously shown to be involved in replication of damaged DNA in somatic cells - is required to protect replication fork integrity in unperturbed ESCs. Our working hypothesis, which will be directly addressed in the next weeks, is that origin firing factors are rate limiting in ESCs, leading to inheritance of partially replicated DNA during fast cell cycle progression. Indeed, overexpression of the firing factor Cdc45 and/or altering the cell cycle length by inhibiting CDK activity, reduces DDR signalling in ESCs. Haematopoietic stem cells (HSCs) possess the ability to give rise to all the cells of the haematopoietic system. HSCs are mainly quiescent and are upon injury or inflammation to bring about tissue homeostasis. Stimulation of mice with interferon alpha (IFN-α) specifically activates dormant HSCs. Preliminary observations in this study suggest that most activated HSCs exhibit elevated γH2AX staining. These results suggest that HSCs that exit from dormancy may experience RS, similarly to actively proliferating ESCs. Collectively, the main findings in this study suggest that the active DDR in proliferating stem cells signals incomplete replication inherited during fast cell cycle progression.