**9. The light entrainment of the circadian clock in zebrafish would reflect a cellular response to photooxidative stress**

It is conceivable that the development of circadian clocks is one way to segregate daytime from nighttime processes with light-dark cycles acting as selective forces

*Oncogenes and Carcinogenesis*

**7. Shared regulatory pathway for circadian clock and DNA repair in** 

*zPHR. The induced zPHR repairs UV-damaged DNA in a light-dependent manner.*

Although solar light has several beneficial uses, including the regulation of circadian clocks, the UV component of solar light is harmful to living cells because it produces cytotoxic and mutagenic lesions in DNA called cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts] (**Figure 2**) [51, 52]. Natural selective pressure has forced the development of a self-defense system mediated by photoreactivation. Photoreactivation is the light-dependent DNA repair mechanism mediated by DNA photolyases (PHRs), which bind to and repair the UV-induced DNA damage using visible light as an energy source [53]. Two classes of PHRs have been identified, one specific for CPDs and the other specific for (6-4) photoproducts. Importantly, both the induction of PHRs in response to light and subsequent light-dependent repair of DNA by PHRs are essential for a successful photoreactivation in zebrafish cells [42, 54, 55]. Notably, the expression of the zebrafish *Phr* repairing (6-4) photoproduct (*z64Phr*) is regulated by the same light-induced MAPK cascades as those controlling the expression of the clock genes *zCry1a* and *zPer2* (**Figure 2**) [49]. The light-induced ERK activation triggers the expression of *z64Phr*, whereas the light-induced p38 activation inhibits it. Thus, the light-dependent DNA repair and regulation of the circadian clock are governed by shared regulatory pathways. Both CRYs and PHRs belong to the DNA photolyase/cryptochrome protein family and have highly similar amino acid sequences [42, 55, 56]. Evolutionary studies have shown that the animal CRY proteins functionally diverged first from the CPD photolyase and then further

*A proposal model of light signaling pathways involved in shared control of the circadian clock and DNA repair in zebrafish. In a variety of organisms, light induces ROS production. In zebrafish cells, the light-induced ROS stimulate intracellular MAPK/ERK signaling pathway, which transduces photic signal to zCry1a expression. The light-induced zCRY1a interacts directly with the zCLOCK:zBMAL complex and modifies its transcriptional capacity. Notably, the zCLOCK:zBMAL complex regulates the transcription of a variety of genes involved in cellular stress responses and DDR. UV component of sunlight induces DNA damage. Light-induced ROS and activation of MAPK/ERK pathway also induce expression of a DNA repair enzyme,* 

**18**

**zebrafish**

**Figure 2.**

[69, 70]. In this scenario, increasing levels of oxygen-free radicals during the daytime may be a decisive factor in relegating the anabolic processes of mitosis, growth, and consolidation to the dark hours. Thus, it is reasonable that cellular signaling cascade mediated by ROS is utilized in the regulation of the circadian clocks and that common regulatory pathways mediate both cellular responses to photooxidative stress and the light-dependent regulation of the circadian clocks (**Figure 2**).

In addition to the photooxidative stress derived from sunlight, the UV component of it is major source of harm to organisms [51, 52]. In zebrafish, the light induces expression of PHRs which repair UV-damaged DNA in a light-dependent manner (**Figure 2**) [49, 71]. Importantly, this light induction of *DNA Phr* expression appears to be mediated by photooxidative stress [68, 72]. These observations are consistent with the idea that photooxidative stress may be utilized as a signal to activate DNA repair enzymes that can protect the organism's DNA from UV-induced damage. The fact that ROS, a well-known inducer of oxidative stress, can activate *zCry1a* transcription in zebrafish cells [66], together with the finding that *zCry1a* and *DNA Phr* are governed by shared light-induced signaling pathways [49], strongly suggests that, at least in zebrafish, the light entrainment of the circadian clock reflects a long-standing cellular response to photooxidative stress (**Figure 2**). The zCRY1a protein interacts directly with the CLOCK (NPAS2):BMAL complexes and regulates its transcriptional capacity [67, 73]. The complexes regulate a variety of key genes involved in cellular stress responses, DNA repair, and cell cycle regulation [14, 74]. Thus, the circadian clock protein zCRY1a may be the key integrator of oxidative stress that controls the core circadian machinery to regulate the transcription of genes responsible for DDR and cell cycle adjustments.
