Christopher P. Selby

Human Transcription-Repair Coupling Factor CSB/ERCC6 Is a DNA-stimulated ATPase but Is Not a Helicase and Does Not Disrupt the Ternary Transcription Complex of Stalled RNA Polymerase II

Transcription is coupled to repair in Escherichia coli and in humans. Proteins encoded by the mfd gene in E. coli and by the ERCC6/CSB gene in humans, both of which possess the so-called helicase motifs, are required for the coupling reaction. It has been shown that the Mfd protein is an ATPase but not a helicase and accomplishes coupling, in part, by disrupting the ternary complex of E. coli RNA polymerase stalled at the site of DNA damage. In this study we overproduced the human CSB protein using the baculovirus vector and purified and characterized the recombinant protein. CSB has an ATPase activity that is stimulated strongly by DNA; however, it neither acts as a helicase nor does it dissociate stalled RNA polymerase II, suggesting a coupling mechanism in humans different from that in prokaryotes. CSB is a DNA-binding protein, and it also binds to XPA, TFIIH, and the p34 subunit of TFIIE. These interactions are likely to play a role in recruiting repair proteins to ternary complexes formed at damage sites.

A cryptochrome/photolyase class of enzymes with single-stranded DNA-specific photolyase activity

Photolyases and cryptochrome blue-light photoreceptors are evolutionarily related flavoproteins that perform distinct functions. Photolyases repair UV-damaged DNA in many species from bacteria to plants and animals. Cryptochromes regulate growth and development in plants and the circadian clock in animals. Recently, a new branch of the photolyase/cryptochrome family was identified. Members of this branch exhibited no or trace levels of DNA repair activity in vivo and in vitro and, therefore, were considered to be cryptochromes, and they were named cryptochrome-DASH. Here, we show that Cry-DASH proteins from bacterial, plant, and animal sources actually are photolyases with high degree of specificity for cyclobutane pyrimidine dimers in ssDNA.

Light induction of a vertebrate clock gene involves signaling through blue-light receptors and MAP kinases.

The signaling pathways that couple light photoreception to entrainment of the circadian clock have yet to be deciphered. Two prominent groups of candidates for the circadian photoreceptors are opsins (e.g., melanopsin) and blue-light photoreceptors (e.g., cryptochromes). We have previously showed that the zebrafish is an ideal model organism in which to study circadian regulation and light response in peripheral tissues. Here, we used the light-responsive zebrafish cell line Z3 to dissect the response of the clock gene zPer2 to light. We show that the MAPK (mitogen-activated protein kinase) pathway is essential for this response, although other signaling pathways may also play a role. Moreover, action spectrum analyses of zPer2 transcriptional response to monochromatic light demonstrate the involvement of a blue-light photoreceptor. The Cry1b and Cry3 cryptochromes constitute attractive candidates as photoreceptors in this setting. Our results establish a link between blue-light photoreceptors, probably cryptochromes, and the MAPK pathway to elicit light-induced transcriptional activation of clock genes.

Control of skin cancer by the circadian rhythm

Skin cancer is the most common form of cancer in the United States. The main cause of this cancer is DNA damage induced by the UV component of sunlight. In humans and mice, UV damage is removed by the nucleotide excision repair system. Here, we report that a rate-limiting subunit of excision repair, the xeroderma pigmentosum group A (XPA) protein, and the excision repair rate exhibit daily rhythmicity in mouse skin, with a minimum in the morning and a maximum in the afternoon/evening. In parallel with the rhythmicity of repair rate, we find that mice exposed to UV radiation (UVR) at 4:00 AM display a decreased latency and about a fivefold increased multiplicity of skin cancer (invasive squamous cell carcinoma) than mice exposed to UVR at 4:00 PM. We conclude that time of day of exposure to UVR is a contributing factor to its carcinogenicity in mice, and possibly in humans.

Reaction mechanism of Drosophila cryptochrome

Cryptochrome (CRY) is a blue-light sensitive flavoprotein that functions as the primary circadian photoreceptor in Drosophila melanogaster. The mechanism by which it transmits the light signal to the core clock circuitry is not known. We conducted in vitro studies on the light-induced conformational change in CRY and its effect on protein–protein interaction and performed in vivo analysis of the lifetime of the signaling state of the protein to gain some insight into the mechanism of phototransduction. We find that exposure of CRY to blue light induces a conformation similar to that of the constitutively active CRY mutant with a C-terminal deletion (CRYΔ). This light-induced conformation has a half-life of ∼15 min in the dark at 25 °C and is characterized by increased affinity to Jetlag E3 ligase. In vivo analysis reveals that in the Drosophila S2 cell line, the signaling state induced by a millisecond light exposure has a half-life of 27 min in the dark at 0 °C during which period it is susceptible to degradation by the ubiquitin-proteasome system. These findings lead to a plausible model for circadian photoreception/phototransduction in Drosophila.

Animal Type 1 Cryptochromes ANALYSIS OF THE REDOX STATE OF THE FLAVIN COFACTOR BY SITE-DIRECTED MUTAGENESIS

It has recently been realized that animal cryptochromes (CRYs) fall into two broad groups. Type 1 CRYs, the prototype of which is the Drosophila CRY, that is known to be a circadian photoreceptor. Type 2 CRYs, the prototypes of which are human CRY 1 and CRY 2, are known to function as core clock proteins. The mechanism of photosignaling by the Type 1 CRYs is not well understood. We recently reported that the flavin cofactor of the Type 1 CRY of the monarch butterfly may be in the form of flavin anion radical, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{FAD}^{{\bar{{\cdot}}}}\) \end{document}, in vivo. Here we describe the purification and characterization of wild-type and mutant forms of Type 1 CRYs from fruit fly, butterfly, mosquito, and silk moth. Cryptochromes from all four sources contain FADox when purified, and the flavin is readily reduced to \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{FAD}^{{\bar{{\cdot}}}}\) \end{document} by light. Interestingly, mutations that block photoreduction in vitro do not affect the photoreceptor activities of these CRYs, but mutations that reduce the stability of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{FAD}^{{\bar{{\cdot}}}}\) \end{document} in vitro abolish the photoreceptor function of Type 1 CRYs in vivo. Collectively, our data provide strong evidence for functional similarities of Type 1 CRYs across insect species and further support the proposal that \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{FAD}^{{\bar{{\cdot}}}}\) \end{document} represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs.

Genome-wide analysis of human global and transcription-coupled excision repair of UV damage at single-nucleotide resolution

We developed a method for genome-wide mapping of DNA excision repair named XR-seq (excision repair sequencing). Human nucleotide excision repair generates two incisions surrounding the site of damage, creating an ∼30-mer. In XR-seq, this fragment is isolated and subjected to high-throughput sequencing. We used XR-seq to produce stranded, nucleotide-resolution maps of repair of two UV-induced DNA damages in human cells: cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts [(6-4)PPs]. In wild-type cells, CPD repair was highly associated with transcription, specifically with the template strand. Experiments in cells defective in either transcription-coupled excision repair or general excision repair isolated the contribution of each pathway to the overall repair pattern and showed that transcription-coupled repair of both photoproducts occurs exclusively on the template strand. XR-seq maps capture transcription-coupled repair at sites of divergent gene promoters and bidirectional enhancer RNA (eRNA) production at enhancers. XR-seq data also uncovered the repair characteristics and novel sequence preferences of CPDs and (6-4)PPs. XR-seq and the resulting repair maps will facilitate studies of the effects of genomic location, chromatin context, transcription, and replication on DNA repair in human cells.

Structure and function of animal cryptochromes

Cryptochrome (CRY) is a photolyase-like flavoprotein with no DNA-repair activity but with known or presumed blue-light receptor function. Animal CRYs have DNA-binding and autokinase activities, and their flavin cofactor is reduced by photoinduced electron transfer. In Drosophila, CRY is a major circadian photoreceptor, and in mammals, the two CRY proteins are core components of the molecular clock and potential circadian photoreceptors. In mammals, CRYs participate in cell cycle regulation and the cellular response to DNA damage by controlling the expression of some cell cycle genes and by directly interacting with checkpoint proteins.

Biochemical Analysis of the Canonical Model for the Mammalian Circadian Clock

The current consensus model for the circadian clock in mammals is based on a transcription-translation feedback loop. In this model, CRY and PER proteins repress their own transcription by suppressing the transactivator function of the CLOCK:BMAL1 heterodimer directly (physical model) and by facilitating post-translational modifications (chemical model). Most of the data for this model come from genetic and cell biological experiments. Here, we have purified all of the core clock proteins and performed in vitro and in vivo biochemical experiments to test the physical model. We find that CLOCK:BMAL1 binds to an E-box sequence in DNA and that CRY binds stably to the CLOCK:BMAL1:E-box ternary complex independently of PER. Both CRY and PER bind to CLOCK and BMAL1 off DNA but, in contrast to CRY, PER does not bind to the CLOCK:BMAL1:E-box complex. Unexpectedly, PER actually interferes with the binding of CRY to the CLOCK:BMAL1:E-box ternary complex. CRY likely destabilizes the CLOCK:BMAL1 heterodimer on DNA by a post-translational mechanism after binding to the complex. These findings support some aspects of the canonical model, but also suggest that some key features of the model need to be revised.

Posttranslational regulation of the mammalian circadian clock by cryptochrome and protein phosphatase 5

The molecular oscillator that drives circadian rhythmicity in mammals obtains its near 24-h periodicity from posttranslational regulation of clock proteins. Activity of the major clock kinase casein kinase I (CKI) ε is regulated by inhibitory autophosphorylation. Here we show that protein phosphatase (PP) 5 regulates the kinase activity of CKIε. We demonstrate that cryptochrome regulates clock protein phosphorylation by modulating the effect of PP5 on CKIε. Like CKIε, PP5 is expressed both in the master circadian clock in the suprachiasmatic nuclei and in peripheral tissues independent of the clock. Expression of a dominant-negative PP5 mutant reduces PER phosphorylation by CKIε in vivo, and down-regulation of PP5 significantly reduces the amplitude of circadian cycling in cultured human fibroblasts. Collectively, these findings indicate that PP5, CKIε, and cryptochrome dynamically regulate the mammalian circadian clock.