Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II (RNAPII) are necessary for DNA damage response (DDR) foci formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNAPII pre-initiation complex (PIC), MED1 and CDK9. Absence or inactivation of these factors causes DDR foci reduction both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins such as 53BP1 into foci that exhibit liquid-liquid phase separation (LLPS) condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis which stimulates phase separation of DDR factors in the shape of foci. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

SUMMARY Excess dormant origins bound by the minichromosome maintenance (MCM) replicative helicase complex play a critical role in preventing replication stress, chromosome instability and tumorigenesis. In response to DNA damage, replicating cells must coordinate DNA repair and dormant origin firing to ensure complete and timely replication of the genome; how cells regulate this process remains elusive. Herein, we identify a member of the Fanconi Anemia (FA) DNA repair pathway, FANCI, as a key effector of dormant origin firing in response to replication stress. Cells lacking FANCI have reduced number of origins, increased inter-origin distances and slowed proliferation rates. Intriguingly, ATR-mediated FANCI phosphorylation inhibits dormant origin firing while promoting replication fork restart/DNA repair. Using super-resolution microscopy, we show that FANCI co-localizes with MCM-bound chromatin in response to replication stress. These data reveal a unique role for FANCI as a modulator of dormant origin firing and links timely genome replication to DNA repair.

No abstract

Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca 2+ transients, increased Ca 2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca 2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca 2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca 2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca 2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca 2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca 2+ , increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca 2+ ] i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca 2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca 2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca 2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

DNA damage-induced signaling by ATR and CHK1 inhibits DNA replication, stabilizes stalled and collapsed replication forks, and mediates the repair of multiple classes of DNA lesions. We and others have shown that ATR kinase inhibitors, three of which are currently undergoing clinical trials, induce excessive origin firing during unperturbed DNA replication, indicating that ATR kinase activity limits replication initiation in the absence of damage. However, the origins impacted and the underlying mechanism(s) have not been described. Here, we show that unperturbed DNA replication is associated with a low level of ATR and CHK1 kinase signaling and that inhibition of this signaling induces dormant origin firing at sites of ongoing replication throughout the S phase. We show that ATR and CHK1 kinase inhibitors induce RIF1 Ser2205 phosphorylation in a CDK1-dependent manner, which disrupts an interaction between RIF1 and PP1 phosphatase. Thus, ATR and CHK1 signaling suppresses CDK1 kinase activity throughout the S phase and stabilizes an interaction between RIF1 and PP1 in replicating cells. PP1 dephosphorylates key CDC7 and CDK2 kinase substrates to inhibit the assembly and activation of the replicative helicase. This mechanism limits origin firing during unperturbed DNA replication in human cells.

DNA base flipping is a fundamental theme in DNA biophysics. The dynamics for a B-DNA base to spontaneously flip out of the double helix has significant implications in various DNA-protein interactions but are still poorly understood. The spontaneous base-flipping rate obtained previously via the imino proton exchange assay is most likely the rate of base wobbling instead of flipping. Using the diffusion-decelerated fluorescence correlation spectroscopy together with molecular dynamics simulations, we show that a base of a single mismatched base pair (T-G, T-T, or T-C) in a doublestranded DNA can spontaneously flip out of the DNA duplex. The extrahelical lifetimes are on the order of 10 ms, whereas the intrahelical lifetimes range from 0.3 to 20 s depending on the stability of the base pairs. These findings provide detailed understanding on the dynamics of DNA base flipping and lay down foundation to fully understand how exactly the repair proteins search and locate the target mismatched base among a vast excess of matched DNA bases.fluctuation spectroscopy | integrated tempering sampling | rate constants | free-energy landscape A base in normal B-DNA spontaneously swinging out of the double helix to an extrahelical position is known as spontaneous base flipping. The dynamics of such base flipping is a fundamental issue in DNA biophysics. It is also related to how DNA repair or modification proteins search and fix the lesion bases to maintain the genome integrity or modify the DNA. Although extensive structural studies have found that many DNA base repair/modification proteins completely flip their target base out extrahelically (so-called enzymatic base flipping) (1-5), it is still under debate (6-11) whether the base flipping occurs spontaneously (9, 10, 12) or not (6-8). Accurate information on the dynamics of spontaneous base flipping is therefore of high interest and importance.However, the study of spontaneous base flipping is deemed to be difficult. The probability is extremely low for a single base to flip out of the DNA double helix in the absence of proteins. Hence only sensitive relaxation methods are able to detect such kind of fluctuation under equilibrium. As a well-known relaxation method, NMR has been applied to tackle this problem through the imino proton exchange assay (9,(13)(14)(15)(16)(17). In this assay, it is assumed that the exchange of the imino proton (in either G or T base) with the catalysts in the solution occurs only when the base flips out (13), and the extrapolated imino proton exchange rate at an infinite catalyst concentration is taken to be the base-flipping rate (14,15,17). According to these NMR studies the lifetime of the extrahelical state is on the order of microseconds, and that of the intrahelical state ranges from milliseconds to hundreds of milliseconds, depending on the stability of individual base pairs. MacKerell and coworkers as well as others have done extensive theoretical investigations and found that the target imino proton on the base already becomes accessible...

The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to ends of DNA double-strand breaks and recruits several factors of the Non-Homologous End Joining (NHEJ) pathway through molecular mechanisms that remain unclear. Here, we describe the crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBMs motifs bind on remote sites of Ku80 α/β domain. The X-KBM occupies an internal pocket formed after an unprecedented large outward rotation of the Ku80 α/β domain. We reveal independent recruitment at laser-irradiated sites of the APLF-interacting protein XRCC4 and of XLF through the respective binding of A- and X-KBMs to Ku80. Finally, we show that mutations on the X-KBM and A KBM binding sites in Ku80 compromises efficiency and accuracy of end-joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchorage points necessary to build the NHEJ intricate interactions network.

DNA hybridization, wherein strands of DNA form duplex or larger hybrids through noncovalent, sequence-specific interactions, is one of the most fundamental processes in biology. Developing a better understanding of the kinetic and dynamic properties of DNA hybridization will thus help in the elucidation of molecular mechanisms involved in numerous biochemical processes. Moreover, because DNA hybridization has been widely adapted in biotechnology, its study is invaluable to the development of a range of commercially important processes. In this Account, we examine recent studies of the kinetics and dynamics of DNA hybridization, including (i) intramolecular collision of random coil, single-stranded DNA (ssDNA), (ii) nucleic acid hairpin folding, and (iii) considerations of DNA hybridization from both a global view and a detailed base-by-base view. We also examine the spontaneous single-base-pair flipping in duplex DNA because of its importance to both DNA hybridization and repair. Intramolecular collision of random coil ssDNA, with chemical relaxation times ranging from hundreds of nanoseconds to a few microseconds, is investigated both theoretically and experimentally. The first passage time theory of Szabo, Schulten, and Schulten, which determines the average reaction time of the intrachain collision, was tested. Although it was found to provide an acceptable approximation, a more sophisticated theoretical treatment is desirable. Nucleic acid hairpin folding has been extensively investigated as an important model system of DNA hybridization. The relaxation time of hairpin folding and unfolding strongly depends on the stem length, and it may range from hundreds of microseconds to hundreds of milliseconds. The traditional two-state model has been revised to a multistate model as a result of new experimental observations and theoretical study, and partially folded intermediate states have been introduced to the folding energy landscape. On the other hand, new techniques are needed to provide more accurate and detailed information on the dynamics of DNA hairpin folding in the time domain of sub-milliseconds to tens of milliseconds. From a global view, the hybridization of unstructured ssDNA goes through an entropy-controlled nucleation step, whereas the hybridization of ssDNA with a hairpin structure must overcome an extra, enthalpy-controlled energy barrier to eliminate the hairpin. From a detailed base-by-base view, however, there exist many intermediate states. The average single-base-pair hybridization and dehybridization rates in a duplex DNA formation have been determined to be on the order of a millisecond. Meanwhile, accurate information on the early stages of hybridization, such as the dynamics of nucleation, is still lacking. The investigation of spontaneous flipping of a single base in a mismatched base pair in a duplex DNA, although very important, has only recently been initiated because of the earlier lack of suitable probing tools. In sum, the study of DNA hybridization offers a rich range of rese...