Mammalian Circadian Clock

The circadian clock is the internal timekeeping system that controls cyclic changes in physiology and behavior to prepare the organism for the unique challenges of the solar day. In mice and humans the circadian rhythm at the organismal level is generated by a molecular clock of periodicity of ~24 hrs. The molecular clock consists of a transcription-translation feedback loop (TTFL) in which the heterodimeric transcriptional activator CLOCK-BMAL1 promotes transcription of transcriptional repressors, CRY (Cryptochrome) and PER (Period) which counter the activity of CLOCK-BMAL1 as shown in Fig. 3A. Our group discovered that CRY is a core human clock protein and that it mediates repression by two mechanisms (Fig. 3B,C): In one CRY binds to the CLOCK-BMAL1 complex on DNA and blocks its interaction with the transcription machinery. In the second mode of repression, the co-repressor PER displaces the entire activator complex from the promoter in a CRY-dependent manner. Our research has provided a mechanistic basic for how this dual repression mechanism confers precision and resilience and, at the same time, flexibility and adaptability to the signaling pathways and networks and therefore influences physio-pathologic conditions ranging from sleep regulation and jetlag to metabolic syndrome to cancer. We discovered that the circadian clock regulates nucleotide excision repair and hence the susceptibility to UV-induced skin cancer as a function of time of the day of exposure to light. Because excision repair is also the main repair mechanism for removing DNA lesions from the genome that are generated by the anti-cancer drug cisplatin, we are currently working at translating our finding of clock-excision repair connection to develop improved chemotherapy regimens.

Control of DNA Repair by the Circadian Clock

We discovered that the circadian clock controls nucleotide excision repair in mammalian organisms. We wish to apply this finding for cancer prevention and treatment because excision repair removes DNA damage caused by carcinogens as well as damage caused by anticancer drugs. We are using mice as the model organism for these studies. We found that excision repair of carcinogenic UV damage in mice is low at early morning hours and reaches its highest level in the evening. As a consequence, mice exposed to carcinogenic UVB light in morning are 4-times more likely to develop invasive skin cancer than mice exposed to the same UVB dose in the evening (Fig. 3A). These findings should have implications for the optimum time for human sun exposure. To determine the effect of the circadian clock on the repair of DNA damage caused by the anticancer drug cisplatin, we injected cisplatin into mice at 4 hour intervals for a period of one day and analyzed the repair of Platinum-DNA adducts over the course of the day genome-wide and at single nucleotide resolution. We found that the repair of Pt-DNA adducts is controlled by two circadian programs in mouse tissue: (1) The clock controls transcription of 1500-2000 genes with expression maxima of different genes spread over the entire day, but with the majority peaking at pre-dawn and pre-dusk. Because transcription stimulates repair of adducts in the transcribed strand (TS), the TS of circadian-controlled genes are repaired at times of day specific for each gene with prominent peaks at pre-dawn and pre-dusk (Fig. 3B). (2) The clock controls expression of the XPA protein and therefore of excision repair activity which peaks at ZT8-10 (ZT=0 is the time when light is turned on, ZT=12 is the time light is turned off under 12h light: 12h dark conditions). Because the basal repair activity has rhythmicity, the repair of Pt-DNA adducts in the regions of the genome that are not transcribed (the nontranscribed strand (NTS) of transcribed genes, both strands of non-transcribed genes, and intergenic regions) peak at ZT8-10. As a consequence of these two circadian programs, in many circadian-controlled genes the peak and trough repair times for TS and NTS are different, and sometimes anti-phase. Cisplatin is the most commonly used chemotherapeutic drug for treating solid tumors. However, the usefulness of this important drug is limited due of its toxicity and primary acquired resistance by cancer cells. Frequently, biochemical pathways of cancer cells are out of synchrony with those in normal tissues, and these are indications that the circadian clock is “broken” in cancerous tissue. We aim to take advantage of our data regarding orderly repair in normal tissue compared to arrhythmic repair in cancer to develop cisplatin administration regiments (Chronochemotherapy) to improve the efficacy and reduce side effects of cisplatin therapy.