Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells.

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-stranded breaks (DSBs) in vertebrates. However, due to challenges in detecting DSBs in living cells, the repair capacity of the NHEJ pathway is unknown. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed ~115Å apart in one of several long-range synaptic complexes before transitioning into a short-range synaptic complex that juxtaposes DNA ends to facilitate ligation. The transition from long-range to short-range synaptic complexes involves both conformational and compositional changes of the NHEJ factors bound to the DNA break. Importantly, it is unclear how NHEJ proceeds in vivo because of the challenges involved in analyzing recruitment of NHEJ factors to DSBs over time in living cells. Here, we develop a new approach to study the temporal and compositional dynamics of NHEJ complexes using live cell single-molecule imaging. Our results provide direct evidence for stepwise maturation of the NHEJ complex, pinpoint key regulatory steps in NHEJ progression, and define the overall repair capacity NHEJ in living cells.

Electron microscopy reveals toroidal shape of master neuronal cell differentiator REST - RE1-silencing transcription factor.

The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.

Transcriptional regulators of human oncoviruses: structural and functional implications for anticancer therapy.

Transcription is often the first biosynthetic event of viral infection. Viruses produce preferentially viral transcriptional regulators (vTRs) essential for expressing viral genes and regulating essential host cell proteins to enable viral genome replication. As vTRs are unique viral proteins that promote the transcription of viral nucleic acid, vTRs interact with host proteins to suppress detection and immune reactions to viral infection. Thus, vTRs are promising therapeutic targets that are sequentially and structurally distinct from host cell proteins. Here, we review vTRs of three human oncoviruses: HBx of hepatitis B virus, HBZ of human T-lymphotropic virus type 1, and Rta of Epstein-Barr virus. We present three cunningly exciting and dangerous transcription strategies that make viral infections so efficient. We use available structural and functional knowledge to critically examine the potential of vTRs as new antiviral-anticancer therapy targets. For each oncovirus, we describe (i) the strategy of viral genome transcription; (ii) vTRs' structure and binding partners essential for transcription regulation; and (iii) advantages and challenges of vTR targeting in antiviral therapies. We discuss the implications of vTR regulation for oncogenesis and perspectives on developing novel antiviral and anticancer strategies.

Quantitative Biology of Human Shelterin and Telomerase: Searching for the Weakest Point.

The repetitive telomeric DNA at chromosome ends is protected from unwanted repair by telomere-associated proteins, which form the shelterin complex in mammals. Recent works have provided new insights into the mechanisms of how human shelterin assembles and recruits telomerase to telomeres. Inhibition of telomerase activity and telomerase recruitment to chromosome ends is a promising target for anticancer therapy. Here, we summarize results of quantitative assessments and newly emerged structural information along with the status of the most promising approaches to telomerase inhibition in cancer cells. We focus on the mechanism of shelterin assembly and the mechanisms of how shelterin affects telomerase recruitment to telomeres, addressing the conceptual dilemma of how shelterin allows telomerase action and regulates other essential processes. We evaluate how the identified critical interactions of telomerase and shelterin might be elucidated in future research of new anticancer strategies.

Human Telomere Repeat Binding Factor TRF1 Replaces TRF2 Bound to Shelterin Core Hub TIN2 when TPP1 Is Absent.

Human telomeric repeat binding factors TRF1 and TRF2 along with TIN2 form the core of the shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1-TIN2-TRF2 complex formation in solution at physiological conditions. Fluorescence cross-correlation spectroscopy was used to describe the complex assembly by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substitutes TRF2 on TIN2. We assessed also the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1-TIN2-TRF2 core complex.