The UVSSA protein is part of a genome integrity homeostasis network with links to transcription-coupled DNA repair and ATM signaling.

SignificanceTranscription-coupled repair (TCR) involves four core proteins: CSA, CSB, USP7, and UVSSA. CSA and CSB are mutated in the severe human neurocutaneous disease Cockayne syndrome. In contrast UVSSA is a mild photosensitive disease in which a mutated protein sequence prevents recruitment of USP7 protease to deubiquitinate and stabilize CSB. We deleted the UVSSA protein using CRISPR-Cas9 in an aneuploid cell line, HEK293, and determined the functional consequences. The knockout cell line was sensitive to transcription-blocking lesions but not sensitive to oxidative agents or PARP inhibitors, unlike CSB. Knockout of UVSSA also activated ATM, like CSB, in transcription-arrested cells. The phenotype of UVSSA, especially its rarity, suggests that many TCR-deficient patients and tumors fail to be recognized clinically.

STRIDE-a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells.

We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. The sensitivity of STRIDE was tested using a specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used terminal deoxynucleotidyl transferase dUTP nick-end labeling assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions) and fragmentation of DNA in human spermatozoa. The STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X-ray repair cross-complementing protein 1 (XRCC1), a key factor in single-strand break and base excision repair, is recruited into nuclear bodies formed in response to replication-related single-strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci. They are structurally organized, containing canonical promyelocytic leukemia (PML) nuclear body protein SP100 concentrated in a peripheral layer, and XRCC1 in the center. They also contain other factors, including PML, poly(ADP-ribose) polymerase 1 (PARP1), ligase IIIα, and origin recognition complex subunit 5. The breast cancer 1 and -2 C terminus domains of XRCC1 are essential for formation of these repair foci. These results reveal that XRCC1-contaning foci constitute newly recognized PML-like nuclear bodies that accrete and locally deliver essential factors for repair of single-strand DNA breaks in replication regions.-Kordon, M. M., Szczurek, A., Berniak, K., Szelest, O., Solarczyk, K., Tworzydlo, M., Wachsmann-Hogiu, S., Vaahtokari, A., Cremer, C., Pederson, T., Dobrucki, J. W. PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

Two stages of XRCC1 recruitment and two classes of XRCC1 foci formed in response to low level DNA damage induced by visible light, or stress triggered by heat shock.

Induction of local photosensitised DNA damage has been used to study recruitment of repair factors, spatial organisation and subsequent stages of the repair processes. However, the damage induced by a focused laser beam interacting with a photosensitiser may not fully reflect the types of damage and repair encountered in cells of an animal under typical conditions in vivo. We report on two characteristic stages of recruitment of XRCC1 (a protein engaged in BER and SSB repair pathways), in response to low level DNA damage induced by visible light. We demonstrate that, when just a few DNA breaks are induced in a small region of the nucleus, the recruited XRCC1 is initially distributed uniformly throughout this region, and rearranges into several small stationary foci within minutes. In contrast, when heavy damage of various types (including oxidative damage) is induced in cells pre-sensitized with a DNA-binding drug ethidium bromide, XRCC1 is also recruited but fails to rearrange from the stage of the uniform distribution to the stage of several small foci, indicating that this heavy damage interferes with the progress and completion of the repair processes. We hypothesize that that first stage may reflect recruitment of XRCC1 to poly(ADP-ribose) moieties in the region surrounding the single-strand break, while the second-binding directly to the DNA lesions. We also show that moderate damage or stress induces formation of two types of XRCC1-containing foci differing in their mobility. A large subset of DNA damage-induced XRCC1 foci is associated with a major component of PML nuclear bodies--the Sp100 protein.

Photosensitized damage inflicted on plasma membranes of live cells by an extracellular generator of singlet oxygen--a linear dependence of a lethal dose on light intensity.

We describe a study of the influence of a dose rate, i.e. light intensity or photon flux, on the efficiency of induction of a loss of integrity of plasma membranes of live cells in culture. The influence of a photon flux on the size of the light dose, which was capable of causing lethal effects, was measured in an experimental system where singlet oxygen was generated exclusively outside of live cells by ruthenium(II) phenantroline complex. Instantaneous, sensitive detection of a loss of integrity of a plasma membrane was achieved by fluorescence confocal imaging of the entry of this complex into a cell interior. We demonstrate that the size of the lethal dose of light is directly proportional to the intensity of the exciting light. Thus, the probability of a photon of the exciting light inflicting photosensitized damage on plasma membranes diminishes with increasing density of the incident photons.

Real-time cell viability assays using a new anthracycline derivative DRAQ7®.

The exclusion of charged fluorescent dyes by intact cells has become a well-established assay for determining viability of cells. In search for a noninvasive fluorescent probe capable of long-term monitoring of cell death in real-time, we evaluated a new anthracycline derivative DRAQ7. The novel probe does not penetrate the plasma membrane of living cells but when the membrane integrity is compromised, it enters and binds readily to nuclear DNA to report cell death. It proved to be nontoxic to a panel of cancer cell lines grown continuously for up to 72 h and did not induce any detectable DNA damage signaling when analyzed using laser scanning microscopy and flow cytometry. The DRAQ7 provided a sensitive, real-time readout of cell death induced by a variety of stressors such as hypoxia, starvation, and drug-induced cytotoxicity. The overall responses to anticancer agents and resulting pharmacological dose-response profiles were not affected by the growth of tumor cells in the presence DRAQ7. Moreover, we for the first time introduced a near real-time microflow cytometric assay based on combination of DRAQ7 and mitochondrial inner membrane potential (ΔΨ(m) ) sensitive probe TMRM. We provide evidence that this low-dosage, real-time labeling procedure provides multiparameter and kinetic fingerprint of anticancer drug action.