119 research outputs found
Mfd Protein and Transcription-Repair Coupling in Escherichia coli
In 1989, transcription-repair coupling (TRC) was first described in Escherichia coli, as the transcription-dependent, preferential nucleotide excision repair (NER) of UV photoproducts located in the template DNA strand. This finding led to pioneering biochemical studies of TRC in the laboratory of Professor Aziz Sancar, where, at the time, major contributions were being made toward understanding the roles of the UvrA, UvrB and UvrC proteins in NER. When the repair studies were extended to TRC, template but not coding strand lesions were found to block RNA polymerase (RNAP) in vitro, and unexpectedly, the blocked RNAP inhibited NER. A transcription-repair coupling factor, also called Mfd protein, was found to remove the blocked RNAP, deliver the repair enzyme to the lesion and thereby mediate more rapid repair of the transcription-blocking lesion compared with lesions elsewhere. Structural and functional analyses of Mfd protein revealed helicase motifs responsible for ATP hydrolysis and DNA binding, and regions that interact with RNAP and UvrA. These and additional studies provided a basis upon which other investigators, in following decades, have characterized fascinating and unexpected structural and mechanistic features of Mfd, revealed the possible existence of additional pathways of TRC and discovered additional roles of Mfd in the cell
The Second Chromophore in Drosophila Photolyase/Cryptochrome Family Photoreceptors
The photolyase/cryptochrome family of proteins are FAD-containing flavoproteins which carry out blue-light dependent functions including DNA repair, plant growth and development, and regulation of the circadian clock. In addition to FAD, many members of the family contain a second chromophore which functions as a photoantenna, harvesting light and transferring the excitation energy to FAD and thus increasing the efficiency of the system. The second chromophore is methenyltetrahydrofolate (MTHF) in most photolyases characterized to date and FAD, FMN, or 5-deazariboflavin in others. To date no second chromophore has been identified in cryptochromes. Drosophila contains 3 members of the cryptochrome/photolyase family: cyclobutane pyrimidine dimer (CPD) photolyase, (6-4) photoproduct photolyase, and cryptochrome. We developed an expression system capable of incorporating all known second chromophores into the cognate cryptochrome/photolyase family members. Using this system we demonstrate that Drosophila CPD photolyase and (6-4) photolyase employ 5-deazariboflavin as their second chromophore but Drosophila cryptochrome, which is evolutionarily closer to (6-4) photolyase than the CPD photolyase, lacks a second chromophore
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
Genome-wide transcription-coupled repair in Escherichia coli is mediated by the Mfd translocase
In transcription-coupled repair (TCR), nucleotide excision repair occurs most rapidly in the template strand of actively transcribed genes. TCR has been observed in a limited set of genes directly assayed in Escherichia coli cells. In vitro, Mfd translocase performs reactions necessary to mediate TCR: It removes RNA polymerase blocked by a template strand lesion and rapidly delivers repair enzymes to the lesion. This study applied excision repair sequencing methodology to map the location of repair sites in different E. coli strains. Results showed that Mfd-dependent TCR is widespread in the E. coli genome. Results with UvrD helicase demonstrated its role in basal repair, but no overall role in TCR
Mammalian Period represses and de-represses transcription by displacing CLOCK–BMAL1 from promoters in a Cryptochrome-dependent manner
The mammalian circadian clock is controlled by a transcription-translation feedback loop consisting of transcriptional activators circadian locomotor output cycles kaput (CLOCK)–brain and muscle Arnt-like protein-1 (BMAL1), which function as a complex at E/E'-box elements, and repressors Cryptochrome 1 (CRY1)/CRY2 and PER1/PER2. CRYs repress upon binding as CRY–CLOCK–BMAL1–E-box complexes. Period proteins (PERs) repress by removing the heterotrimeric complexes from the E-box. We report here that in the Cry1 promoter, the CRY1–CLOCK–BMAL1–E-box complex represses a transcriptional activator acting in cis, and removal of the heterotrimeric complex by PER2 de-represses the transcriptional activator. ChIP-seq and RNA-seq experiments identified other genes also de-repressed by PER2. These data clarify the role of PER2 and reveal the level of complexity in regulation of Cry1 and other circadian-controlled genes
Mechanism of Photosignaling by Drosophila Cryptochrome: ROLE OF THE REDOX STATUS OF THE FLAVIN CHROMOPHORE
Cryptochrome (CRY) is the primary circadian photoreceptor in Drosophila. Upon light absorption, dCRY undergoes a conformational change that enables it to bind to Timeless (dTIM), as well as to two different E3 ligases that ubiquitylate dTIM and dCRY, respectively, resulting in their proteolysis and resetting the phase of the circadian rhythm. Purified dCRY contains oxidized flavin (FADox), which is readily photoreduced to the anionic semiquinone through a set of 3 highly conserved Trp residues (Trp triad). The crystal structure of dCRY has revealed a fourth Trp (Trp-536) as a potential electron donor. Previously, we reported that the Trp triad played no role in photoinduced proteolysis of dCRY in Drosophila cells. Here we investigated the role of the Trp triad and Trp-536, and the redox status of the flavin on light-induced proteolysis of both dCRY and dTIM and resetting of the clock. We found that both oxidized (FADox) and reduced (FAD⨪) forms of dCRY undergo light-induced conformational change in vitro that enable dCRY to bind JET and that Trp triad and Trp-536 mutations that block known or presumed intraprotein electron transfer reactions do not affect dCRY phototransduction under bright or dim light in vivo as measured by light-induced proteolysis of dCRY and dTIM in Drosophila S2R+ cells. We conclude that both oxidized and reduced forms of dCRY are capable of photosignaling
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, FAD(*-), 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 FAD(ox) when purified, and the flavin is readily reduced to FAD(*-) 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 FAD(*-) 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 FAD(*-) represents the ground state and not the excited state of the flavin cofactor in Type 1 CRYs
Comparative Photochemistry of Animal Type 1 and Type 4 Cryptochromesâ€
Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities
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
Human Transcription Release Factor 2 Dissociates RNA Polymerases I and II Stalled at a Cyclobutane Thymine Dimer
RNA polymerase II stalled at a lesion in the transcribed strand is thought to constitute a signal for transcription-coupled repair. Transcription factors that act on RNA polymerase in elongation mode potentially influence this mode of repair. Previously, it was shown that transcription elongation factors TFIIS and Cockayne's syndrome complementation group B protein did not disrupt the ternary complex of RNA polymerase II stalled at a thymine cyclobutane dimer, nor did they enable RNA polymerase II to bypass the dimer. Here we investigated the effect of the transcription factor 2 on RNA polymerase II and RNA polymerase I stalled at thymine dimers. Transcription factor 2 is known to release transcripts from RNA polymerase II early elongation complex generated by pulse-transcription. We found that factor 2 (which is also called release factor) disrupts the ternary complex of RNA polymerase II at a thymine dimer and surprisingly exerts the same effect on RNA polymerase I. These findings show that in mammalian cells a RNA polymerase I or RNA polymerase II transcript truncated by a lesion in the template strand may be discarded unless repair is accomplished rapidly by a mechanism that does not displace stalled RNA polymerases
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