Abstract
DNA replication fork stalling or collapse that arises from endogenous damage poses a serious threat to genome stability, but cells invoke an intricate signaling cascade referred to as the DNA damage response (DDR) to prevent such damage. The gene product ataxia telangiectasia and Rad3-related (ATR) responds primarily to replication stress by regulating cell cycle checkpoint control, yet it’s role in DNA repair, particularly homologous recombination (HR), remains unclear. This is of particular interest since HR is one way in which replication restart can occur in the presence of a stalled or collapsed fork. Hypomorphic mutations in human ATR cause the rare autosomal-recessive disease Seckel syndrome, and complete loss of Atr in mice leads to embryonic lethality. We recently adapted the in vivo murine pink-eyed unstable (pun) assay for measuring HR frequency to be able to investigate the role of essential genes on HR using a conditional Cre/loxP system. Our system allows for the unique opportunity to test the effect of ATR loss on HR in somatic cells under physiological conditions. Using this system, we provide evidence that retinal pigment epithelium (RPE) cells lacking ATR have decreased density with abnormal morphology, a decreased frequency of HR and an increased level of chromosomal damage.
Highlights
DNA damage is an unavoidable consequence of life resulting from both endogenous and exogenous sources
We recently developed a conditional system to assess the role of essential genes on homologous recombination (HR) frequency by excising the gene of interest only in the retinal pigment epithelium (RPE) [24]
Expression of the Cre transgene is driven by the tyrosinase related protein 1 (Trp1) promoter, whose expression is restricted to the RPE during mouse embryonic development [32]
Summary
DNA damage is an unavoidable consequence of life resulting from both endogenous and exogenous sources. Without an appropriate response, such interruptions to DNA replication can lead to genome instability. To ensure that chromosomes are accurately and faithfully duplicated, cells have evolved an elaborate set of DDR mechanisms in which DNA replication slows allowing for the recruitment of DNA repair factors while preventing potentially deleterious progression through cell cycle [1]. One such response to replication stress involves the protein kinase ATR. Activation of ATR by replication-blocking DNA damage elicits a pleiotropic signal transduction pathway that includes numerous transducer and effector proteins [2]
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