A potential role for RNA aminoacylation prior to its role in peptide synthesis.

Coded ribosomal peptide synthesis could not have evolved unless its sequence and amino acid-specific aminoacylated tRNA substrates already existed. We therefore wondered whether aminoacylated RNAs might have served some primordial function prior to their role in protein synthesis. Here, we show that specific RNA sequences can be nonenzymatically aminoacylated and ligated to produce amino acid-bridged stem-loop RNAs. We used deep sequencing to identify RNAs that undergo highly efficient glycine aminoacylation followed by loop-closing ligation. The crystal structure of one such glycine-bridged RNA hairpin reveals a compact internally stabilized structure with the same eponymous T-loop architecture that is found in many noncoding RNAs, including the modern tRNA. We demonstrate that the T-loop-assisted amino acid bridging of RNA oligonucleotides enables the rapid template-free assembly of a chimeric version of an aminoacyl-RNA synthetase ribozyme. We suggest that the primordial assembly of amino acid-bridged chimeric ribozymes provides a direct and facile route for the covalent incorporation of amino acids into RNA. A greater functionality of covalently incorporated amino acids could contribute to enhanced ribozyme catalysis, providing a driving force for the evolution of sequence and amino acid-specific aminoacyl-RNA synthetase ribozymes in the RNA World. The synthesis of specifically aminoacylated RNAs, an unlikely prospect for nonenzymatic reactions but a likely one for ribozymes, could have set the stage for the subsequent evolution of coded protein synthesis.

Shelterin-Mediated Telomere Protection.

For more than a decade, it has been known that mammalian cells use shelterin to protect chromosome ends. Much progress has been made on the mechanism by which shelterin prevents telomeres from inadvertently activating DNA damage signaling and double-strand break (DSB) repair pathways. Shelterin averts activation of three DNA damage response enzymes [the ataxia-telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases and poly(ADP-ribose) polymerase 1 (PARP1)], blocks three DSB repair pathways [classical nonhomologous end joining (c-NHEJ), alternative (alt)-NHEJ, and homology-directed repair (HDR)], and prevents hyper-resection at telomeres. For several of these functions, mechanistic insights have emerged. In addition, much has been learned about how shelterin maintains the telomeric 3' overhang, forms and protects the t-loop structure, and promotes replication through telomeres. These studies revealed that shelterin is compartmentalized, with individual subunits dedicated to distinct aspects of the end-protection problem. This review focuses on the current knowledge of shelterin-mediated telomere protection, highlights differences between human and mouse shelterin, and discusses some of the questions that remain.

The DDR at telomeres lacking intact shelterin does not require substantial chromatin decompaction.

Telomeres are protected by shelterin, a six-subunit protein complex that represses the DNA damage response (DDR) at chromosome ends. Extensive data suggest that TRF2 in shelterin remodels telomeres into the t-loop structure, thereby hiding telomere ends from double-stranded break repair and ATM signaling, whereas POT1 represses ATR signaling by excluding RPA. An alternative protection mechanism was suggested recently by which shelterin subunits TRF1, TRF2, and TIN2 mediate telomeric chromatin compaction, which was proposed to minimize access of DDR factors. We performed superresolution imaging of telomeres in mouse cells after conditional deletion of TRF1, TRF2, or both, the latter of which results in the complete loss of shelterin. Upon removal of TRF1 or TRF2, we observed only minor changes in the telomere volume in most of our experiments. Upon codeletion of TRF1 and TRF2, the telomere volume increased by varying amounts, but even those samples exhibiting small changes in telomere volume showed DDR at nearly all telomeres. Upon shelterin removal, telomeres underwent 53BP1-dependent clustering, potentially explaining at least in part the apparent increase in telomere volume. Furthermore, chromatin accessibility, as determined by ATAC-seq (assay for transposase-accessible chromatin [ATAC] with high-throughput sequencing), was not substantially altered by shelterin removal. These results suggest that the DDR induced by shelterin removal does not require substantial telomere decompaction.

Position and root resorption of the incisors following anterior segment retraction using friction versus frictionless mechanics: A randomised controlled trial.

AIM: To evaluate the three-dimensional position and root resorption of incisors after anterior segment retraction (ASR) using friction versus frictionless mechanics. PARTICIPANTS AND METHODS: Thirty female patients (13-18 years) with bimaxillary protrusion were randomly allocated into two groups. In the intervention group, ASR was undertaken using an elastomeric chain rendering 160 g/side extending between mini-screw implant and a hook crimped on 0.017 × 0.025-inch stainless-steel wire distal to the lateral incisor. In the comparison group, ASR was undertaken using closing T-loops fabricated from 0.017 × 0.025-inch titanium molybdenum alloy (TMA) wire rendering comparable retraction force. In both groups, the canine brackets were ligated after retraction to the mini-screw implants that were inserted in both the upper and lower arches bilaterally. The primary outcome was the three-dimensional changes in the position of the incisors. The secondary outcome was root resorption. These were measured from cone-beam computed tomography scans. RESULTS: Statistically significant decreases in the upper (UI) and lower incisors (LI) crown torque were seen in both groups; however, the difference between groups was not statistically or clinically significant (UI MD -2.04°; 95% confidence interval [CI] = -8.02-3.95; LI MD -0.49°; 95% CI = -7.06-6.08). Significant tipping of upper (MD -1.17°; 95% CI = -2.06--0.27) and lower (MD -1.13°; 95% CI = -1.66--0.60) incisors was found in the friction, but not the frictionless group after retraction; however, the changes were not clinically significant. Significant lower incisor intrusion was found in both groups after retraction; however, the difference between groups was not statistically or clinically significant (MD -0.61°; 95% CI = -1.99-0.77). Statistically significant decreases in the UI and LI root length were seen in both groups. The difference between groups for UI changes was statistically significant (MD 0.54 mm; 95% CI = -0.02-1.07) but probably not clinically significant. CONCLUSION: Considering the limitations in the current study, there was no advantage of either mechanics over the other regarding the final position of incisors. The likelihood of root resorption should be considered when frictionless mechanics are used for retraction of incisors. REGISTRY: Clinicaltrials.gov (NCT04878939).

Crystal structures of an unmodified bacterial tRNA reveal intrinsic structural flexibility and plasticity as general properties of unbound tRNAs.

Ubiquitous across all domains of life, tRNAs constitute an essential component of cellular physiology, carry out an indispensable role in protein synthesis, and have been historically the subject of a wide range of biochemical and biophysical studies as prototypical folded RNA molecules. Although conformational flexibility is a well-established characteristic of tRNA structure, it is typically regarded as an adaptive property exhibited in response to an inducing event, such as the binding of a tRNA synthetase or the accommodation of an aminoacyl-tRNA into the ribosome. In this study, we present crystallographic data of a tRNA molecule to expand on this paradigm by showing that structural flexibility and plasticity are intrinsic properties of tRNAs, apparent even in the absence of other factors. Based on two closely related conformations observed within the same crystal, we posit that unbound tRNAs by themselves are flexible and dynamic molecules. Furthermore, we demonstrate that the formation of the T-loop conformation by the tRNA TΨC stem-loop, a well-characterized and classic RNA structural motif, is possible even in the absence of important interactions observed in fully folded tRNAs.

Establishment, FEM analysis and experimental validation of tooth movement prediction model of orthodontic archwire T-loop.

BACKGROUND: The T-loop has been used clinically to close gap between teeth. And it is a typical orthodontic archwire bending method. However, the design of the T-loop parameters for different patients is based on the clinical experience of the dentists. The variation in dentists' clinical experience is the main reason for inadequate orthodontic treatment, even high incidence of postoperative complications. METHODS: Firstly, the tooth movement prediction model is established based on the analysis of the T-loop structure and the waxy model dynamic resistance. As well as the reverse reconstruction of the complete maxillary 3D model based on the patient CBCT images, the oral biomechanical FEM analysis is completed. A maxillary waxy dental model is manufactured to realize the water-bath measurement experiment in vitro mimicking the oral bio-environment. Thus, the calculated, simulation and experimental data are obtained, as well as obtaining a cloud of total deformation from the simulation analysis. RESULTS: The growth trend of the 11 sets of simulation data is the same as that of the experimental data. And all of them show that the tooth displacement is positively correlated with the cross-sectional size of the archwire, and the clearance distance. As well as the higher Young's modulus of the archwire material, the greater the tooth displacement. And the effect of archwire parameters on tooth displacement derived from simulation and experimental data is consistent with the prediction model. The experimental and calculated data are also compared and analyzed, and the two kinds of data are basically consistent in terms of growth trends and fluctuations, with deviation rates ranging from 2.17 to 10.00%. CONCLUSIONS: This study shows that the accuracy and reliability of the tooth movement prediction model can be verified through the comparative analysis and deviation calculation of the obtained calculated, simulation and experimental data, which can assist dentists to safely and efficiently perform orthodontic treatment on patients. And the FEM analysis can achieve predictability of orthodontic treatment results.

The Connection Between Cell Fate and Telomere.

Abolition of telomerase activity results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death. Telomere shortening leads to the attainment of the "Hayflick limit", and the transition of cells to state of senescence. If senescence is bypassed, cells undergo crisis through loss of checkpoints. This process causes massive cell death concomitant with further telomere shortening and spontaneous telomere fusions. In functional telomere of mammalian cells, DNA contains double-stranded tandem repeats of TTAGGG. The Shelterin complex, which is composed of six different proteins, is required for the regulation of telomere length and stability in cells. Telomere protection by telomeric repeat binding protein 2 (TRF2) is dependent on DNA damage response (DDR) inhibition via formation of T-loop structures. Many protein kinases contribute to the DDR activated cell cycle checkpoint pathways, and prevent DNA replication until damaged DNA is repaired. Thereby, the connection between cell fate and telomere length-associated telomerase activity is regulated by multiple protein kinase activities. Contrarily, inactivation of DNA damage checkpoint protein kinases in senescent cells can restore cell-cycle progression into S phase. Therefore, telomere-initiated senescence is a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres. In this review, in addition to the above mentioned, the choice of main repair pathways, which comprise non-homologous end joining and homologous recombination in telomere uncapping telomere dysfunctions, are discussed.

Replicating through telomeres: a means to an end.

Proper replication of the telomeric DNA at chromosome ends is critical for preserving genome integrity. Yet, telomeres present challenges for the replication machinery, such as their repetitive and heterochromatic nature and their potential to form non-Watson-Crick structures as well as the fact that they are transcribed. Numerous telomere-bound proteins are required to facilitate progression of the replication fork throughout telomeric DNA. In particular, shelterin plays crucial functions in telomere length regulation, protection of telomeres from nuclease degradation, control of DNA damage response at telomeres, and the recruitment of associated factors required for telomere DNA processing and replication. In this review we discuss the recently uncovered functions of mammalian telomere-specific and telomere-associated proteins that facilitate proper telomere replication.

What I got wrong about shelterin.

The ASBMB 2018 Bert and Natalie Vallee award in Biomedical Sciences honors our work on shelterin, a protein complex that helps cells distinguish the chromosome ends from sites of DNA damage. Shelterin protects telomeres from all aspects of the DNA damage response, including ATM and ATR serine/threonine kinase signaling and several forms of double-strand break repair. Today, this six-subunit protein complex could easily be identified in one single proteomics step. But, it took us more than 15 years to piece together the entire shelterin complex, one protein at a time. Although we did a lot of things right, here I tell the story of shelterin's discovery with an emphasis on the things that I got wrong along the way.

Analysis of reversed L-loops as closing loops with anterior intrusive force.

OBJECTIVE: Intrusive forces on anterior brackets are preferable for avoiding overbite deepening. Reversing plain L-loops may create such advantageous force system during space closure. DESIGN: Force systems of reversed L-loops were compared with T-loops at three interbracket distances (IBD). SETTING: Computational study. METHODS: Using finite element analysis, loop response during simulated loop-pulling was determined for plain reversed L- and T-loop configurations at three IBDs and two sizes. Force systems were calculated on both loop ends for two activation forces. RESULTS: The 12 mm IBD reversed L-loops had almost equal M/F ratios in opposite directions at both ends. A small intrusive force was found at the canine bracket (CB). The 6 mm IBD reversed L-loops showed larger M/F ratios and extrusive forces at the premolar bracket (PB) and smaller M/F with intrusive forces at CB. The force system of 12 mm IBD T-loops showed the similar force systems as off-centered V-bends with extrusive force at CB, whilst plain 6 mm IBD T-loops showed properties similar to centered V-bends with less extrusive force at CB. CONCLUSIONS: Reversed L closing loops placed no extrusive force on the CB end at various IBDs, indicating that reversed loops will generate an intrusive force at anterior teeth during space closure.

Evaluation of Biomechanical Properties of Four Loops at Different Activation: A Finite Element Method Study.

AIM: The aim of the study was to evaluate the force, moment, and moment/force ratio (M/F) generated by activating T loop, Kalra Simultaneous Intrusion and Retraction (KSIR) loop, Omega loop, and Teardrop loop made of titanium molybdenum alloy (TMA) wire with different preactivation bends at 1, 2, and 4 mm activation. MATERIALS AND METHODS: Finite element method (FEM) models of the four loops were created and different preactivation bends were placed. The loops were then activated and analyzed for force, moment, and M/F ratio using ANSYS software. RESULTS: In loops without preactivation bends, highest force values were generated by Omega loop, whereas T loop had the least force value. The mean value for the M/F in the alpha segment was almost similar. In loops with preactivation bend, the force was highest in Teardrop loop, whereas T loop had the least force value. The mean value for the M/F in the alpha segment was almost similar in all the loops. CONCLUSION: T loop with preactivation bend shows the most favorable properties. CLINICAL SIGNIFICANCE: T loop is comparatively reliable for the frictionless mechanics for the space closure than the other loops evaluated in clinical use.

Principles of ion recognition in RNA: insights from the group II intron structures.

Metal ions promote both RNA folding and catalysis, thus being essential in stabilizing the structure and determining the function of large RNA molecules, including group II introns. The latter are self-splicing metalloribozymes, containing a heteronuclear four-metal-ion center within the active site. In addition to these catalytic ions, group II introns bind many other structural ions, including delocalized ions that bind the RNA diffusively and well-ordered ions that bind the RNA tightly with high occupancy. The latter ions, which can be studied by biophysical methods, have not yet been analyzed systematically. Here, we compare crystal structures of the group IIC intron from Oceanobacillus iheyensis and classify numerous site-bound ions, which are primarily localized in the intron core and near long-range tertiary contacts. Certain ion-binding sites resemble motifs observed in known RNA structures, while others are idiosyncratic to the group II intron. Particularly interesting are (1) ions proximal to the active site, which may participate in splicing together with the catalytic four-metal-ion center, (2) organic ions that bind regions predicted to interact with intron-encoded proteins, and (3) unusual monovalent ions bound to GU wobble pairs, GA mismatches, the S-turn, the tetraloop-receptor, and the T-loop. Our analysis extends the general principles by which ions participate in RNA structural organization and it will aid in the determination and interpretation of future RNA structures.

Structural basis of Cdk7 activation by dual T-loop phosphorylation.

Cyclin-dependent kinase 7 (Cdk7) occupies a central position in cell-cycle and transcriptional regulation owing to its function as both a CDK-activating kinase (CAK) and part of the general transcription factor TFIIH. Cdk7 forms an active complex upon association with Cyclin H and Mat1, and its catalytic activity is regulated by two phosphorylations in the activation segment (T loop): the canonical activating modification at T170 and another at S164. Here we report the crystal structure of the fully activated human Cdk7/Cyclin H/Mat1 complex containing both T-loop phosphorylations. Whereas pT170 coordinates a set of basic residues conserved in other CDKs, pS164 nucleates an arginine network involving all three subunits that is unique to the ternary Cdk7 complex. We identify differential dependencies of kinase activity and substrate recognition on individual phosphorylations within the Cdk7 T loop. The CAK function of Cdk7 is not affected by T-loop phosphorylation, whereas activity towards non-CDK substrates is increased several-fold by phosphorylation at T170. Moreover, dual T-loop phosphorylation at both T170 and S164 stimulates multi-site phosphorylation of transcriptional substrates-the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and the SPT5 carboxy-terminal repeat (CTR) region. In human cells, Cdk7-regulatory phosphorylation is a two-step process in which phosphorylation of S164 precedes, and may prime, T170 phosphorylation. Thus, dual T-loop phosphorylation can regulate Cdk7 through multiple mechanisms, with pS164 supporting tripartite complex formation and possibly influencing Cdk7 processivity, while the canonical pT170 enhances kinase activity towards critical substrates involved in transcription.

Sequence, structure, and stacking: specifics of tRNA anchoring to the T box riboswitch.

The term riboswitch usually refers to small molecule sensing regulatory modules in the 5' untranslated regions of a mRNA. They are typically comprised of separate ligand binding and regulatory domains. The T box riboswitch is unique from other identified riboswitches because its effector is an essential macromolecule, tRNA. It senses the aminoacylation state of tRNA to regulate genes involved in a variety of functions relating to amino acid metabolism and tRNA aminoacylation. T box riboswitches performs an intuitively simple process using a complex structured RNA element and, until recently, the underlying mechanisms were poorly understood. Only two sequence-specific contacts had been previously identified: (1) between the specifier sequence (codon) and the tRNA anticodon and (2) between an anti-terminator stem loop and the tRNA acceptor arm CCA tail. tRNA aminoacylation blocks the latter interaction and therefore serves as the switch between termination and anti-termination. Outside of these two contacts, the structure and functions of T box riboswitches have come to light in some recent studies. We recently described the X-ray crystal structure of the highly conserved T box riboswitch distal Stem I region and demonstrated that this region interacts with the tRNA elbow to anchor it to the riboswitch. Independently, Lehmann et al. used sequence homology search to arrive at a similar model for Stem I-tRNA interactions. The model was further supported by two recent structures of the Stem I-tRNA complex, determined independently by our group and by Zhang and Ferré-D'Amaré. This article highlights some of these contributions to synthesize an updated model for tRNA recognition by the T box riboswitch.

Evolution of transient RNA structure-RNA polymerase interactions in respiratory RNA virus genomes.

RNA viruses are important human pathogens that cause seasonal epidemics and occasional pandemics. Examples are influenza A viruses (IAV) and coronaviruses (CoV). When emerging IAV and CoV spill over to humans, they adapt to evade immune responses and optimize their replication and spread in human cells. In IAV, adaptation occurs in all viral proteins, including the viral ribonucleoprotein (RNP) complex. RNPs consist of a copy of the viral RNA polymerase, a double-helical coil of nucleoprotein, and one of the eight segments of the IAV RNA genome. The RNA segments and their transcripts are partially structured to coordinate the packaging of the viral genome and modulate viral mRNA translation. In addition, RNA structures can affect the efficiency of viral RNA synthesis and the activation of host innate immune response. Here, we investigated if RNA structures that modulate IAV replication processivity, so-called template loops (t-loops), vary during the adaptation of pandemic and emerging IAV to humans. Using cell culture-based replication assays and in silico sequence analyses, we find that the sensitivity of the IAV H3N2 RNA polymerase to t-loops increased between isolates from 1968 and 2017, whereas the total free energy of t-loops in the IAV H3N2 genome was reduced. This reduction is particularly prominent in the PB1 gene. In H1N1 IAV, we find two separate reductions in t-loop free energy, one following the 1918 pandemic and one following the 2009 pandemic. No destabilization of t-loops is observed in the influenza B virus genome, whereas analysis of SARS-CoV-2 isolates reveals destabilization of viral RNA structures. Overall, we propose that a loss of free energy in the RNA genome of emerging respiratory RNA viruses may contribute to the adaption of these viruses to the human population.

Context-dependence of T-loop Mediated Long-range RNA Tertiary Interactions.

The architecture and folding of complex RNAs is governed by a limited set of highly recurrent structural motifs that form long-range tertiary interactions. One of these motifs is the T-loop, which was first identified in tRNA but is broadly distributed across biological RNAs. While the T-loop has been examined in detail in different biological contexts, the various receptors that it interacts with are not as well defined. In this study, we use a cell-based genetic screen in concert with bioinformatic analysis to examine three different, but related, T-loop receptor motifs found in the flavin mononucleotide (FMN) and cobalamin (Cbl) riboswitches. As a host for different T-loop receptors, we employed the env8 class-II Cbl riboswitch, an RNA that uses two T-loop motifs for both folding and supporting the ligand binding pocket. A set of libraries was created in which select nucleotides that participate in the T-loop/T-loop receptor (TL/TLR) interaction were fully randomized. Library members were screened for their ability to support Cbl-dependent expression of a reporter gene. While T-loops appear to be variable in sequence, we find that the functional sequence space is more restricted in the Cbl riboswitch, suggesting that TL/TLR interactions are context dependent. Our data reveal clear sequence signatures for the different types of receptor motifs that align with phylogenic analysis of these motifs in the FMN and Cbl riboswitches. Finally, our data suggest the functional contribution of various nucleobase-mediated long-range interactions within the riboswitch subclass of TL/TLR interactions that are distinct from those found in other RNAs.

Comparison of Maxillary Canine Retraction Using Split-Mouth Design With Dual Force Cuspid Retractor and T-loop Segmental Arch: A Split-Mouth Randomized Clinical Trial.

Introduction This study was designed to explore the differences between two frictionless mechanics for canine retraction i.e., dual force cuspid retractor and T-loop segmental arch. T-loop for canine retraction creates a biomechanical system to deliver a predetermined force and a relatively constant moment-to-force ratio whereas dual force cuspid retractor uses power arms on buccal as well as palatal aspects for canine retraction. Bodily tooth movement can be achieved by both methods, but in this study, our main focus was to reduce the canine retraction timing with better three-dimensional control. Method This split-mouth study was conducted on a total of 20 cuspids of ten patients (five male and five female). Where one side of the arch was selected for T-loop and the other side for dual force cuspid retractor, randomly. Inclusion criteria for this study were; no congenitally missing teeth (excluding third molar), class I or class II molar relationship, no previous history of orthodontic treatment, good oral periodontal status, patients in whom extraction of maxillary first premolar during treatment was indicated. Both groups were compared for the duration of canine retraction, anchorage loss; tipping, and rotation of cuspid and molar, individually, after retraction. Result The result of this study showed that the duration of canine retraction was significantly less in group one, i.e., dual force cuspid retractor 73.8 ± 12.38 days, than in group two, i.e., T-loop 109.4 ± 16.71 days. The anchorage loss in group one was 0.60 ± 0.61 mm and that in group two was 2.40 ± 0.87 mm. Also, the amount of tipping and rotation of the cuspid and molar individually was significantly lesser in group one than in group two. Conclusion In this study, the dual force cuspid retractor shortens the duration of canine retraction with better three-dimensional control and better anchorage preservation when compared to T-loop.

Unveiling the noncanonical activation mechanism of CDKs: insights from recent structural studies.

The Cyclin-dependent kinases (CDKs) play crucial roles in a range of essential cellular processes. While the classical two-step activation mechanism is generally applicable to cell cycle-related CDKs, both CDK7 and CDK8, involved in transcriptional regulation, adopt distinct mechanisms for kinase activation. In both cases, binding to their respective cyclin partners results in only partial activity, while their full activation requires the presence of an additional subunit. Recent structural studies of these two noncanonical kinases have provided unprecedented insights into their activation mechanisms, enabling us to understand how the third subunit coordinates the T-loop stabilization and enhances kinase activity. In this review, we summarize the structure and function of CDK7 and CDK8 within their respective functional complexes, while also describing their noncanonical activation mechanisms. These insights open new avenues for targeted drug discovery and potential therapeutic interventions in various diseases related to CDK7 and CDK8.

Negative and ambisense RNA virus ribonucleocapsids: more than protective armor.

SUMMARYNegative and ambisense RNA viruses are the causative agents of important human diseases such as influenza, measles, Lassa fever, and Ebola hemorrhagic fever. The viral genome of these RNA viruses consists of one or more single-stranded RNA molecules that are encapsidated by viral nucleocapsid proteins to form a ribonucleoprotein complex (RNP). This RNP acts as protection, as a scaffold for RNA folding, and as the context for viral replication and transcription by a viral RNA polymerase. However, the roles of the viral nucleoproteins extend beyond these functions during the viral infection cycle. Recent advances in structural biology techniques and analysis methods have provided new insights into the formation, function, dynamics, and evolution of negative sense virus nucleocapsid proteins, as well as the role that they play in host innate immune responses against viral infection. In this review, we discuss the various roles of nucleocapsid proteins, both in the context of RNPs and in RNA-free states, as well as the open questions that remain.

A ROS-dependent mechanism promotes CDK2 phosphorylation to drive progression through S phase.

Reactive oxygen species (ROS) at the right concentration promote cell proliferation in cell culture, stem cells, and model organisms. However, the mystery of how ROS signaling is coordinated with cell cycle progression and integrated into the cell cycle control machinery on the molecular level remains unsolved. Here, we report increasing levels of mitochondrial ROS during the cell cycle in human cell lines that target cyclin-dependent kinase 2 (CDK2). Chemical and metabolic interferences with ROS production decrease T-loop phosphorylation on CDK2 and so impede its full activation and thus its efficient DNA replication. ROS regulate CDK2 activity through the oxidation of a conserved cysteine residue near the T-loop, which prevents the binding of the T-loop phosphatase KAP. Together, our data reveal how mitochondrial metabolism is coupled with DNA replication and cell cycle progression via ROS, thereby demonstrating how KAP activity toward CDKs can be cell cycle regulated.