Folding kinetics of recognition loop peptides from a photolyase and cryptochrome-DASH

Base flipping, the conformational change of a nucleobase to an extrahelical position, is a key step in the enzymatic repair of damaged DNA. An assay that can detect the flipped-out species in free solution without covalent modification of the DNA would be desirable. The design and synthesis of a simple, sensitive, and rapid assay using specific noncovalent binding to pyrimidines by zinc-cyclen and a commonly used fluorescent reporter group, dansyl, is reported. The binding of the zinc-cyclen unit to a flipped-out thymine base results in a change in the fluorescent properties of the dansyl group that is distinct from nonspecific binding to duplex DNA or intercalation into either the flipped-in or flipped-out species. The assay was tested using fluorescence spectroscopy and detection at 533 +/- 5 nm with normal and abasic duplex DNA as negative and positive controls. The data obtained are fitted to a one-site binding model to determine the equilibrium constant for the two-step process involving base flipping and binding to be approximately 10-6 M.

DNA base flipping is an important mechanism in molecular enzymology, but its study is limited by the lack of an accessible and reliable diagnostic technique. A series of crystalline complexes of a DNA methyltransferase, M.HhaI, and its cognate DNA, in which a fluorescent nucleobase analogue, 2-aminopurine (AP), occupies defined positions with respect the target flipped base, have been prepared and their structures determined at higher than 2 Å resolution. From time-resolved fluorescence measurements of these single crystals, we have established that the fluorescence decay function of AP shows a pronounced, characteristic response to base flipping: the loss of the very short (∼100 ps) decay component and the large increase in the amplitude of the long (∼10 ns) component. When AP is positioned at sites other than the target site, this response is not seen. Most significantly, we have shown that the same clear response is apparent when M.HhaI complexes with DNA in solution, giving an unambiguous signal of base flipping. Analysis of the AP fluorescence decay function reveals conformational heterogeneity in the DNA–enzyme complexes that cannot be discerned from the present X-ray structures.

Background: Disruptions in circadian rhythm have been implicated in prostate cancer (PCa) biology. Previous studies have shown that the core circadian regulatory cryptochrome 1, CRY1, is pro-tumorigenic and associated with poor outcome in PCa. Furthermore, metabolic reprogramming is a hallmark of oncogenic progression, promoting tumor growth through nutrient and energy imbalance. While evidence links circadian rhythm disruptions to metabolic disorders, the underlying mechanism(s) connecting circadian dysregulation and metabolic rewiring in PCa remain poorly understood. Our preliminary data suggest that CRY1 modulates metabolic homeostasis in both preclinical and clinical models of PCa. This study investigates CRY1 as a crucial regulator of PCa growth and survival through metabolic rewiring, aiming to identify novel therapeutic vulnerabilities in CRY1-driven disease. Methods: To elucidate the role of CRY1 in metabolic regulation and disease progression, we utilized hormone therapy-sensitive (HTS) and castration-resistant prostate cancer (CRPC) models with both pharmacological and genetic perturbations of CRY1. Multiomic analyses, including bulk RNA-seq, steady-state metabolomics, and a metabolic focused CRISPR loss-of-function screen, were performed to define CRY1-dependent transcriptional and metabolic programs driving PCa progression. Functional validation studies are being conducted in vitro and ex vivo to assess the impact of CRY1 inhibition on tumor growth and metabolic adaptation. Results: Integrated multiomic analyses revealed that CRY1 drives significant metabolic alterations in bile acid metabolism, epithelial–mesenchymal transition (EMT), apoptosis, and inflammatory in HTS models, while lipid metabolism and cell proliferation Pathways were significantly altered in CRPC models. Furthermore, both CRY1 knockdown and pharmacological inhibition suppressed cell growth in vitro and in vivo, underscoring the importance of CRY1 in maintaining metabolic fitness and tumor viability. Overlapping targets identified across transcriptomic, metabolomic, and CRISPR screening datasets are undergoing biochemical validation. Ongoing drug response studies using CRY1 inhibitor, alone or in combination with metabolic inhibitors, are being conducted in preclinical models and patients derived explants (PDEs) to better recapitulate tumor microenvironmental context and therapeutic response. These studies aim to uncover synergistic vulnerabilities that can be leveraged for precision medicine approaches. Conclusions: Collectively, our findings demonstrate that CRY1 promotes tumor growth by sustaining metabolic homeostasis in PCa. Inhibition of CRY1 disrupts this metabolic balance, impairing tumor viability. Thus, these results support the development of combinatorial therapeutic strategies targeting circadian and metabolic dependencies to improve PCa patient outcome. Citation Format: Arwa Fallatah, Stefan DiFazio, Lakshmi Ravindranath, Ayesha Shafi. Discern the Impact of Cryptochrome 1 (CRY1) on Metabolic Rewiring in PCa [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr B019.

The RepA protein from bacteriophage P1 binds DNA to initiate replication. RepA covers one face of the DNA and the binding site has a completely conserved T that directly faces RepA from the minor groove at position +7. Although all four bases can be distinguished through contacts in the major groove of B-form DNA, contacts in the minor groove cannot easily distinguish between A and T bases. Therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching into the minor groove of B-form DNA. RepA binding sites with modified base pairs at position +7 were used to investigate contacts with RepA. The data show that RepA contacts the N3 proton of T at position +7 and that the T=A hydrogen bonds are already broken in the DNA before RepA binds. To accommodate the N3 proton contact the T(+7 )/A(+7)((')) base pair must be distorted. One possibility is that T(+7) is flipped out of the helix. The energetics of the contact allows RepA to distinguish between all four bases, accounting for the observed high sequence conservation. After protein binding, base pair distortion or base flipping could initiate DNA melting as the second step in DNA replication.

Cryptochromes: sensory reception, transduction, and clock functions subserving circadian systems

The EcoRV DNA methyltransferase introduces a CH3 group at the 6-amino position of the first dA in the duplex sequence d(GATATC). It has previously been reported that the methylase contacts the four phosphates (pNpNpGpA) at, and preceding, the 5'-end of the recognition sequence as well as the single dG in this sequence (Szczelkun, M. D., Jones, H., and Connolly, B. A. (1995) Biochemistry 34, 10734-10743). To study the possible role of the dA and T bases within the ATAT sequence, interference studies have been carried out using diethylpyrocarbonate and osmium tetroxide. The methylase bound very strongly to hemimethylated oligonucleotides modified at the second AT, of the ATAT sequence, in the unmethylated strand of the duplex. This probably arises because these modifications facilitate DNA distortion that follows the binding of the nucleic acid to the protein. Oligonucleotides containing modified bases at both the target dA base and its complementary T were used to determine whether this dA methylase flips out its target base in a similar manner to that observed for dC DNA methylases. In binary EcoRV methylase-DNA complexes, analogues that weakened the base pair caused an increase in affinity between the protein and the nucleic acid. In contrast, in ternary EcoRV methylase-DNA-sinefungin (an analogue of the natural co-factor, S-adenosyl-L-methionine (AdoMet)) complexes, only small differences in affinity were observed between the normal dA-T base pair and the analogues. These results are almost identical to those seen with DNA dC methylases (Klimasauskas, S., and Roberts R. J. (1995) Nucleic Acid Res. 23, 1388-1395; Yang, S. A., Jiang-Cheng, S., Zingg, J. M., Mi, S., and Jones, P. A. (1995) Nucleic Acids Res. 23, 1380-1387) and support a base-flipping mechanism for DNA dA methylases.

Circadian rhythms are endogenous autonomous 24-h oscillations that are generated by a transcription-translation feedback loop (TTFL). In the positive arm of the TTFL, two transcription factors activate the expression of two genes of the negative arm as well as circadian clock-regulated genes. The circadian clocks are reset through photoreceptor proteins by sunlight in the early morning to keep synchrony with the geological clock. Among animal circadian photoreceptors, Drosophila Cryptochrome (DmCRY) has some unique properties because Drosophila has a single cryptochrome (CRY) that appears to have functions which are specific to organs or tissues, or even to a subset of cells. In mammals, CRYs are not photoreceptors but function in the TTFL, while insects have a light-insensitive mammalian-like CRY or a Drosophila-like photoreceptor CRY (or both). Here, we postulate that as being just one CRY in Drosophila, DmCRY might play different roles in different tissues/organs in a context-dependent manner. In addition to being a circadian photoreceptor/protein, attributing also a magnetoreception function to DmCRY has increased its workload. Considering that DmCRY senses photons as a photoreceptor but also can regulate many different events in a light-dependent manner, differential protein-protein interactions (PPIs) of DmCRY might play a critical role in the generation of such diverse outputs. Therefore, we need to add novel approaches in addition to the current ones to study multiple and context-dependent functions of DmCRY by adopting recently developed techniques. Successful identification of transient/fast PPIs on a scale of minutes would enhance our understanding of light-dependent and/or magnetoreception-associated reactions.

The circadian rhythm in glucocorticoid secretion by the adrenal cortex was long considered to be a product and direct reflection of the circadian rhythm of ACTH secretion by the anterior pituitary. However, data have accumulated for several years indicating that changes in circulating ACTH cannot fully explain the adrenal rhythm, and under many conditions there is a dissociation between ACTH and glucocorticoids (1). The circadian rhythm in plasma glucocorticoid concentration is extremely robust, and in many species has a much greater amplitude than the ACTH rhythm (2). In rats after hypophysectomy, the corticosteroid rhythm persists and exhibits 3-fold differences in response to exogenous ACTH, depending on the time of day (3, 4); and in capuchin monkeys, markedly rhythmical cortisol secretion continues after ACTH suppression with dexamethasone (5). Additional findings of nycthemeral differences in adrenal responsiveness to ACTH (6, 7), the ability of a restricted feeding schedule to alter those rhythms (8), and the demonstration that innervation of the adrenal cortex via the splanchnic nerve plays a major role in regulation of circadian glucocorticoid secretion (2, 9) have all contributed to our understanding that stimulation of adrenal steroidogenesis is subject to a number of potent influences in addition to ACTH. Circadian clocks, both the central rhythm generator in the suprachiasmatic nuclei of the hypothalamus and the peripheral oscillators that have been discovered in multiple mammalian tissues, regulate temporal output via interlocking transcriptional/ translational feedback loops involving the core clock genes Per1-2, Cry1-2, Clock, and Bmal1, and their protein products (10). In recent years the molecular machinery necessary for generation of circadian rhythms has been found in the adrenal cortex of rodents and primates, and the adrenal clock of capuchin monkeys maintains a circadian rhythm in vitro (11). These findings suggest that this molecular adrenal clock may also be among the modulators of the steroidogenic response to ACTH. In this issue of Endocrinology, Torres-Farfan et al. (12) have demonstrated that in the capuchin monkey (Cebus apella) adrenal cortex, an antisense knockdown of the expression of the adrenal clock protein cryptochrome 2 (CRY2) blocks the adrenal’s steroidogenic response to stimulation by ACTH. Another recent study showed that expressionofadifferent canonical clockprotein, BMAL1, is necessary for steroidogenesis in the Y-1 immortalized mouse adrenocortical cell line (13). In addition, a transgenic mouse line in which a part of the BMAL1 coding region was expressed in an antisense orientation under tissue-specific control failed to express rhythms of corticosteroid synthesis and secretion when the animals were maintained in constant darkness. Because steroidogenic cells store very little steroid, acute increases in secretion in response to a stimulus require rapid synthesis of new steroid, and the rate-limiting step of transfer of cholesterol across the mitochondrial membranes is dependent on de novo protein synthesis (14). Acute steroidogenesis was found to be accompanied by the synthesis of a phosphoprotein (15) that occurs much more rapidly than ACTH-elicited synthesis of steroidogenic enzymes. The protein was cloned and named steroidogenic acute regulatory protein (StAR) (16). In the aforementioned studies in which adrenal steroidogenic responses to ACTH were found to be dependent on clock proteins and in several other investigations of factors observed to modulate steroidogenesis, the effects were achieved at least partly by alteration of StAR transcription. StAR is expressed only in steroidogenic tissues [adrenal cortex, testes, ovaries, placenta, and brain (17)] and mediates the transfer of mobilized cholesterol from the outer to the inner mitochondrial membrane, the site of the first enzymatic step in steroidogenesis, side chain cleavage of cholesterol to form pregnenolone. The clinical consequence of dysfunctional StARs resulting from gene mutations is congenital lipoid adrenal hyperplasia (18). The absence of functional StAR results in a roughly 85% loss of steroidogenesis that elicits compensatory ACTH secretion and subsequent cellular damage from massive accumulation in lipid droplets of esterified cholesterol that cannot be transported into mitochondria (14).

Background: Impairment of the circadian clock has been associated with numerous diseases, including sleep disorders and metabolic disease. Although small molecules that modulate clock function may form the basis of drug discovery of clock-related diseases, only a few compounds that selectively target core clock proteins have been identified. Three scaffolds were previously discovered as small-molecule activators of the clock protein Cryptochrome (CRY), and they have been providing powerful tools to understand and control the circadian clock system. Identifying new scaffolds will expand the possibilities of drug discovery. Methods: A methylbenzimidazole derivative TH401 identified from cell-based circadian screens was characterized. Effects of TH401 on circadian rhythms were evaluated in cellular assays. Functional assays and X-ray crystallography were used to elucidate the effects of the compound on CRY1 and CRY2 isoforms. Results: TH401 lengthened the period of circadian rhythms and stabilized both CRY1 and CRY2. The compound repressed Per2 reporter activity, which was reduced by Cry1 or Cry2 knockout and abolished by Cry1/Cry2 double knockout, indicating the dependence on CRY isoforms. Thermal shift assays showed slightly higher interaction of TH401 with CRY2 over CRY1. The crystal structure of CRY1 in complex with TH401 revealed a conformational change of the gatekeeper W399, which is involved in isoform-selectivity determination. Conclusions: The present study identified a new small molecule TH401 that targets both CRY isoforms. This compound has expanded the chemical diversity of CRY activators, and will ultimately aid in the development of therapeutics against circadian clock-related disorders.

The circadian rhythm is a fundamental process that regulates the sleep–wake cycle. This rhythm is regulated by core clock genes that oscillate to create a physiological rhythm of circadian neuronal activity. However, we do not know much about the mechanism by which circadian inputs influence neurons involved in sleep–wake architecture. One possible mechanism involves the photoreceptor cryptochrome (CRY). In Drosophila, CRY is receptive to blue light and resets the circadian rhythm. CRY also influences membrane potential dynamics that regulate neural activity of circadian clock neurons in Drosophila, including the temporal structure in sequences of spikes, by interacting with subunits of the voltage-dependent potassium channel. Moreover, several core clock molecules interact with voltage-dependent/independent channels, channel-binding protein, and subunits of the electrogenic ion pump. These components cooperatively regulate mechanisms that translate circadian photoreception and the timing of clock genes into changes in membrane excitability, such as neural firing activity and polarization sensitivity. In clock neurons expressing CRY, these mechanisms also influence synaptic plasticity. In this review, we propose that membrane potential dynamics created by circadian photoreception and core clock molecules are critical for generating the set point of synaptic plasticity that depend on neural coding. In this way, membrane potential dynamics drive formation of baseline sleep architecture, light-driven arousal, and memory processing. We also discuss the machinery that coordinates membrane excitability in circadian networks found in Drosophila, and we compare this machinery to that found in mammalian systems. Based on this body of work, we propose future studies that can better delineate how neural codes impact molecular/cellular signaling and contribute to sleep, memory processing, and neurological disorders.

In Drosophila circadian rhythms persist in constant darkness (DD). The small ventral Lateral Neurons (s-LNv) mainly control the behavioral circadian rhythm in consortium with the large ventral Lateral Neurons (l-LNv) and dorsal Lateral Neurons (LNd). It is believed that the molecular oscillations of clock genes are the source of this persistent behavior. Indeed the s-LNv, LNd, Dorsal Neurons (DN)-DN2 and DN3 displayed self-sustained molecular oscillations in DD both at RNA and protein levels, except the DN2 oscillates in anti-phase. In contrast, the l-LNv and DN1 displayed self-sustained oscillations at the RNA level, but protein oscillations quickly dampened. Having self-sustained and dampened molecular oscillators together in the DN groups suggested that they play different roles. However, all DN groups seemed to contribute together to the light–dark (LD) behavioral rhythm. The LD entrainment of LN oscillators is achieved through Rhodopsin (RH) and Cryptochrome (CRY). CRY's expression in all DN groups implicates also its role in LD entrainment of DN, like in DN1. However, mutations in cry and glass that did not inflict LD synchronization of the DN2, DN3 oscillator implicate the existence of a novel photoreceptor at least in DN3.

DNA methyltransferases play crucial roles in establishing and maintenance of DNA methylation, which is an important epigenetic mark. Flipping the target cytosine out of the DNA helical stack and into the active site of protein provides DNA methyltransferases with an opportunity to access and modify the genetic information hidden in DNA. To investigate the conversion process of base flipping in the HhaI methyltransferase (M.HhaI), we performed different molecular simulation approaches on M.HhaI-DNA-S-adenosylhomocysteine ternary complex. The results demonstrate that the nonspecific binding of DNA to M.HhaI is initially induced by electrostatic interactions. Differences in chemical environment between the major and minor grooves determine the orientation of DNA. Gln237 at the target recognition loop recognizes the GCGC base pair from the major groove side by hydrogen bonds. In addition, catalytic loop motion is a key factor during this process. Our study indicates that base flipping is likely to be an “induced-fit” process. This study provides a solid foundation for future studies on the discovery and development of mechanism-based DNA methyltransferases regulators.

Circadian disruption influences metabolic health. Metabolism modulates circadian function. However, the mechanisms coupling circadian rhythms and metabolism remain poorly understood. Here, we report that cystathionine β-synthase (CBS), a central enzyme in one-carbon metabolism, functionally interacts with the core circadian protein cryptochrome 1 (CRY1). In cells, CBS augments CRY1-mediated repression of the CLOCK/BMAL1 complex and shortens circadian period. Notably, we find that mutant CBS-I278T protein, the most common cause of homocystinuria, does not bind CRY1 or regulate its repressor activity. Transgenic CbsZn/Zn mice, while maintaining circadian locomotor activity period, exhibit reduced circadian power and increased expression of E-BOX outputs. CBS function is reciprocally influenced by CRY1 binding. CRY1 modulates enzymatic activity of the CBS. Liver extracts from Cry1-/- mice show reduced CBS activity that normalizes after the addition of exogenous wild-type (WT) CRY1. Metabolomic analysis of WT, CbsZn/Zn , Cry1-/- , and Cry2-/- samples highlights the metabolic importance of endogenous CRY1. We observed temporal variation in one-carbon and transsulfuration pathways attributable to CRY1-induced CBSactivation. CBS-CRY1 binding provides a post-translational switch to modulate cellular circadian physiology and metabolic control.

Background: Disruption of natural light cycles, as experienced by shift workers, is linked to enhanced cancer incidence. Several mouse models of cancer develop more severe disease when exposed to irregular light/dark cycles, supporting the connection between circadian disruption and increased cancer risk. Cryptochrome 2 (CRY2), a repressive component of the molecular circadian clock, facilitates turnover of the oncoprotein c-MYC, one mechanism that may link the molecular clock to tumorigenesis. In Eμ-MYC mice, which express transgenic c-MYC in B cells and develop aggressive lymphomas and leukemia, global Cry2 deletion reduces survival and enhances tumor formation. Lighting conditions that mimic the disruption experienced by shift workers dampen Cry2 transcripts in peripheral tissues of C57BL/6J mice. Although it is milder than homozygous deletion of Cry2, we hypothesized that reduced Cry2 rhythmicity could alter MYC protein accumulation and contribute to enhanced cancer risk caused by circadian disruption. We tested this hypothesis in MYC-driven lymphoma. Methods: We housed Eμ-MYC mice in light-tight boxes set to either control (continuous cycles of 12-hours of light followed by 12-hours of dark, LD12:12) or chronic jetlag (eight-hour light phase advances every two to three days, CJL) lighting conditions and assessed the impact of disrupted light cycles on survival and tumor formation in Eμ-MYC mice. Results: Environmental disruption of circadian rhythms did not alter tumor location, tumor growth, or survival in Eμ-MYC mice. Conclusions: Dampened rhythms of Cry2 following disruption of circadian light exposures is milder than deletion of Cry2. The lack of phenotype caused by altered circadian gene expression in contrast to enhanced tumorigenesis caused by homozygous deletion of Cry2 suggests that CRY2 dosage impacts this model. Importantly, these findings indicate that increased cancer risk associated with circadian disruption arises from one or more mechanisms that are not recapitulated here, and may be different in distinct tumor types.

Mammalian-type CRYPTOCHROME (CRY-m) is considered to be a core repressive component of the circadian clock in various insect species. However, this role is based only on the molecular function of CRY-m in cultured cells and it therefore remains unknown whether CRY-m is indispensable for governing physiological rhythms at the organismal level. In the present study, we show that RNA interference (RNAi) targeting of cry-m in the bean bug Riptortus pedestris disrupts the circadian clock governing the cuticle deposition rhythm and results in the generation of a single cuticle layer. Furthermore, period expression was induced in cry-m RNAi insects. These results verified that CRY-m functions as a negative regulator in the circadian clock that generates physiological rhythm at the organismal level.