**3. Possible roles of clock proteins in functional regulation of crucial components of DDR pathways**

The activities associated with the physiological processes are organized in daily manner: during the daytime, the animal's physiology is given over to the catabolic processes, whereas at night, it concentrates on the anabolic functions of growth, repair, and consolidation [5, 6]. Disrupt, the time-dependent regulation of physiological functions in animals has profound effects on their health. In particular, many studies have provided evidence that disruption of the circadian clocks results

*Oncogenes and Carcinogenesis*

time cues. The second component is the input which refers to the pathway through which these cues are perceived and act upon the central pacemaker. The third element applies to how the clock affects physiology, which is achieved through the output pathways. In vertebrates, the cellular clocks are comprised of the circadian locomotor output cycles kaput (CLOCK), neuronal PAS domain-containing protein 2 (NPAS2), brain and muscle arnt-like protein-1 (BMAL), period (PER), and cryptochrome (CRY) proteins (**Figure 1A**) [11]. CLOCK or NPAS2 heterodimerize with BMAL to form an active transcription complex that transactivates clock-controlled genes, including *Cry* and *Per*. Once the CRY and PER proteins have been translated, they are translocated to the nucleus, where they inhibit CLOCK(NPAS2):BMALmediated transcription through a direct protein-protein interaction. Importantly,

*Molecular mechanisms establishing circadian clocks in vertebrates. (A) Model of the vertebrate cellular clocks. Two basic helix-loop-helix PAS domain-containing transcription factors CLOCK and BMAL constitute the positive elements. When these transcription factors heterodimerize, they bind to E-boxes to drive the transcription of the negative components of the clock, Per and Cry genes. The products of these clock genes then negatively regulate their own expression, setting up the rhythmic oscillations of gene expression that drive the circadian clocks. CLOCK:BMAL complex also regulates clock-controlled genes, whose products mediate the "output" function of the clocks. CK1 phosphorylates PER protein, which is required for ubiquitination of PER and its subsequent degradation. An essential prerequisite for the circadian feedback loop is a short half-life of clock proteins. Thus, CK1-mediated degradation of PER is critical for maintenance of circadian rhythmicity of* 

*cellular clock. (B) Schematic representation of the proteins that are acetylated by CLOCK protein.*

**14**

**Figure 1.**

in tumorigenesis [8, 9]. Importantly, mice with mutations in the *Bmal1* gene show premature aging phenotype [15]. In addition, human CLOCK has been suggested to be involved in metastasis of colorectal cancer [16]. These findings implicate the core circadian machinery in the regulation of DDR and the cell cycle. Indeed, the circadian regulators have been demonstrated to interact with crucial components of cellular stress response pathways including the ATM, the checkpoint kinase 2 (Chk2) kinase [17], sirtuin1 (SIRT1) deacetylase [18], and nuclear receptors 19, 20], whereas it has been reported that DNA damage can act as a resetting cue for the mammalian circadian clock [21].

Histone acetyltransferases (HATs) such as CBP/p300 are known to acetylate nonhistone targets and have also been recognized as tumor suppressors [22, 23]. Translocation, amplification, overexpression, or mutation of HAT genes are known to occur in several forms of cancer, and several key cell cycle proteins (including p53 and c-MYC) are known targets of HATs. These observations suggest that HATs can also affect cell proliferation and differentiation in multiple ways, in addition to chromatin remodeling. It was previously reported that a core circadian regulator, CLOCK, has intrinsic HAT activity [24] and further that it acetylates a nonhistone target, the heterodimeric CLOCK-binding partner BMAL (**Figure 1B**) [25]. CLOCK also acetylates the glucocorticoid receptor and the argininosuccinate synthase, negatively regulating the transactivation capacity and the enzymatic activity, respectively [20, 26]. It is conceivable that CLOCK would directly interact with and regulate key DDR regulators, leading to the acetylation of these proteins and thereby modulating their activities (**Figure 1B**).
