The Homologous Recombination Machinery Orchestrates Post-replication DNA Repair During Self-renewal of Mouse Embryonic Stem Cells

Abstract

Embryonic stem (ES) cells require homologous recombination (HR) to cope with genomic instability caused during self-renewal. Here, we report expression dynamics and localization of endogenous HR factors in DNA break repair of ES cells. In addition, we analyzed gene expression patterns of HR-related factors at the transcript level with RNA-sequencing experiments. We showed that ES cells constitutively expressed diverse HR proteins throughout the cell cycle and that HR protein expression was not significantly changed even in the DNA damaging conditions. We further analyzed that depleting Rad51 resulted in the accumulation of larger single-stranded DNA (ssDNA) gaps, but did not perturb DNA replication, indicating that ES cells were able to enter the G2-phase in the presence of unrepaired DNA gaps, consistent with the possibility that post-replication repair helps avoid stalling at the G2/M checkpoint. Interestingly, caffeine treatment inhibited the formation of Rad51 or Rad54 foci, but not the formation of γH2AX and Exo1 foci, which led to incomplete HR in ssDNA, thus increasing DNA damage sensitivity. Our results suggested that ES cells possess conserved HR-promoting machinery to ensure effective recruitment of the HR proteins to DNA breaks, thereby driving proper chromosome duplication and cell cycle progression in ES cells.

E2F1 facilitates DNA break repair by localizing to break sites and enhancing the expression of homologous recombination factors

“…More precisely, regulation of the recombinases RAD51, RAD52, and RAD54 has been shown to affect genomic integrity and regulation of the cell cycle. Our results demonstrated that cells lacking RAD51 experienced cell cycle arrest in the G2/M phase and the accumulation of DNA breaks, apoptosis, and unrepaired ssDNA, implying that the HR factor-dependent pathway is likely essential for the maintenance of genomic integrity 35 .…”

“…The transcription factor E2F1 is involved in DNA replication and repair in many types of human cancers 38 . Previous studies have reported that E2F1 is expressed at high levels in most human cancers, including colon cancer and breast cancer and that E2F1 expression in an individual cell facilitates the efficient repair of DSBs and stalled replication forks 35,39 . However, the reason for high-level E2F1 expression in human cancer cells and the specific role of E2F1 in maintaining genomic integrity in cancerous cells are not clearly understood.…”

“…2d, e). Thus, aberrant replication caused by the absence of E2F1 leads to an increase in the number of DNA gaps, which results in activation of the G2/M checkpoint 35 .…”

ATP-binding cassette protein ABCF1 couples gene transcription with maintenance of genome integrity in embryonic stem cells

“…Even though ES cells lack a robust innate immune system, they are nonetheless hypersensitive to DNA damage and readily undergo differentiation and apoptosis ( 85 – 87 ). This is not due to a diminished DNA repair capacity because many repair-related factors are in fact elevated in ES cells ( 88 – 90 ). Taken together, these observations suggest that alternative mechanisms likely exist in order to sensitize ES cells to these cellular insults, thereby protecting the pristine state of ES cells from infections and genome instability.…”

BRPF3-HUWE1-mediated regulation of MYST2 is required for differentiation and cell-cycle progression in embryonic stem cells

“…In addition, because Myst2 accelerates cell proliferation [17], we checked the function of Myst2 on cell-cycle progression in mESCs. The transition from G1 phase to S phase was blocked by treatment with double thymidine and then the cell-cycle progression was released as previously described [18] (Fig. 7c).…”