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Photolyase and cryptochrome belong to a group of structurally similar flavoproteins but with two distinct functions of DNA repair as a photoenzyme and signal transduction as a photoreceptor, respectively, under blue-light illumination. Here, we studied a recently discovered bifunctional Chlamydomonas reinhardtii cryptochrome (CraCRY) with focus on its repair of UV-induced pyrimidine-pyrimidone (6-4) photoproduct (6-4PP). We used femtosecond spectroscopy and site-directed mutagenesis to map out the critical elementary steps by following the dynamics of initial reactants, various intermediates, and final products. We observed initial direct ultrafast electron tunneling from the hydroquinone flavin cofactor to 6-4PP in 300 ps through an intervening adenine as a mediator, minimizing the electron bifurcation of a two-step hopping pathway bridged also by the adenine as an intermediate. The subsequent proton transfer (PT) from the neighboring histidine to anionic 6-4PP in 2 ns is critical and competes with the futile back electron transfer in 151 ps. Mutations of either of two histidines in the active site nearly abolish repair, indicating their essential role on repair reactivity and structural integrity. These results elucidate the electron-coupled PT mechanism and the repair photocycle of this bifunctional cryptochrome at the molecular level, further supporting that the hydroquinone flavin is the active state in vivo for dual functions or one of the two functions is excessive. [ABSTRACT FROM AUTHOR]

DNA repair processes modulate genotoxicity, mutagenesis, and adaption. Nucleotide excision repair removes bulky DNA damage, and in Escherichia coli, basal excision repair, carried out by UvrA, B, C, and D, with DNA PolI and DNA ligase, occurs genome-wide. In transcription-coupled repair (TCR), the Mfd protein targets template strand (TS) lesions that block RNA polymerase for accelerated repair by the basal repair enzymes. Accelerated repair is also seen with particular adducts. Notably, of the two major UV photoproducts, basal repair of (6-4) photoproducts [(6-4) PPs] is about 10× faster than repair of cyclobutane pyrimidine dimers (CPDs). To better understand repair prioritization in E. coli, we used XR-seq to measure TCR of UV photoproducts genome-wide. With CPDs, we found that TCR occurred at early time points, increased with transcription level, and was Mfd dependent; later, with completion of TS repair, nontranscribed strand (NTS) repair predominated. With (6-4) PP, when analyzing all genes, TCR was not observed; in fact, among the most highly transcribed genes, slightly more repair of (6-4) PPs in the NTS was evident. Thus, the very rapid basal repair of (6-4) PP in the NTS was faster than TCR of (6-4) PPs in the TS. Overall, TCR is of limited importance in (6-4) PP repair, and TCR of CPDs is limited to the TS of more highly transcribed genes. These results are consistent with the significant role of Mfd in mutagenesis and the modest effect of mfd deletion on UV survival and bear upon the response of E. coli to bulky DNA damage. [ABSTRACT FROM AUTHOR]

Synechococcus elongatus is an important cyanobacterium that serves as a versatile and robust model for studying circadian biology and photosynthetic metabolism. Its transcriptional regulatory network (TRN) is of fundamental interest, as it orchestrates the cell's adaptation to the environment, including its response to sunlight. Despite the previous characterization of constituent parts of the S. elongatus TRN, a comprehensive layout of its topology remains to be established. Here, we decomposed a compendium of 300 high-quality RNA sequencing datasets of the model strain PCC 7942 using independent component analysis. We obtained 57 independently modulated gene sets, or iModulons, that explain 67% of the variance in the transcriptional response and 1) accurately reflect the activity of known transcriptional regulations, 2) capture functional components of photosynthesis, 3) provide hypotheses for regulon structures and functional annotations of poorly characterized genes, and 4) describe the transcriptional shifts under dynamic light conditions. This transcriptome-wide analysis of S. elongatus provides a quantitative reconstruction of the TRN and presents a knowledge base that can guide future investigations. Our systems-level analysis also provides a global TRN structure for S. elongatus PCC 7942. [ABSTRACT FROM AUTHOR]

One important goal of circadian medicine is to apply time-of-day dosing to improve the efficacy of chemotherapy. However, limited knowledge of how the circadian clock regulates DNA repair presents a challenge to mechanism-based clinical application. We studied time-series genome-wide nucleotide excision repair in liver and kidney of wild type and three different clock mutant genotypes (Cry1-/-Cry2-/-, Per1-/-Per2-/-, and Bmal1-/-). Rhythmic repair on the nontranscribed strand was lost in all three clock mutants. Conversely, rhythmic repair of hundreds of genes on the transcribed strand (TSs) persisted in the livers of Cry1-/-Cry2-/- and Per1-/-Per2-/- mice. We identified a tissue-specific, promoter element-driven repair mode on TSs of collagen and angiogenesis genes in the absence of clock activators or repressors. Furthermore, repair on TSs of thousands of genes was altered when the circadian clock is disrupted. These data contribute to a better understanding of the regulatory role of the circadian clock on nucleotide excision repair in mammals and may be invaluable toward the design of time-aware platinum-based interventions in cancer. [ABSTRACT FROM AUTHOR]

The XR-seq (eXcision Repair-sequencing) method has been extensively used to map nucleotide excision repair genome-wide in organisms ranging from Escherichia coli to yeast, Drosophila, Arabidopsis, mice, and humans. The basic feature of the method is to capture the excised oligomers carrying DNA damage, sequence them, and align their sequences to the genome. We wished to perform XR-seq in vitro with cell-free extract supplemented with a damaged DNA substrate so as to have greater flexibility in investigating factors that affect nucleotide excision repair in the cellular context [M. J. Smerdon, J. J. Wyrick, S. Delaney, J. Biol. Chem. 299, 105118 (2023)]. We report here the successful use of ultraviolet light-irradiated plasmids as substrates for repair in vitro and in vivo by E. coli and E. coli cell-free extracts and by mammalian cell-free extract. XR-seq analyses demonstrated common excision product length and sequence characteristics in vitro and in vivo for both the bacterial and mammalian systems. This approach is expected to help understand the effects of epigenetics and other cellular factors and conditions on DNA repair. [ABSTRACT FROM AUTHOR]