Myosin VI regulates the spatial organisation of mammalian transcription initiation.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.

First passage time study of DNA strand displacement.

DNA strand displacement, in which a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule fluorescence resonance energy transfer approach termed the "fission" assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nucleotide displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence, except for T → U substitution. However, displacement times were largely insensitive to the monovalent salt concentration in the range of 0.25-1 M. Using a one-dimensional random walk model, we infer that the single-step displacement time is in the range of âˆ¼30-300 µs, depending on the base identity. The framework presented here is broadly applicable to the kinetic analysis of multistep processes investigated at the single-molecule level.

A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9.

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models.

DNA damage alters nuclear mechanics through chromatin reorganization.

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

The roles of nuclear myosin in the DNA damage response.

Myosin within the nucleus has often been overlooked due to their importance in cytoplasmic processes and a lack of investigation. However, more recently, it has been shown that their nuclear roles are just as fundamental to cell function and survival with roles in transcription, DNA damage and viral replication. Myosins can act as molecular transporters and anchors that rely on their actin binding and ATPase capabilities. Their roles within the DNA damage response can varies from a transcriptional response, moving chromatin and stabilizing chromosome contacts. This review aims to highlight their key roles in the DNA damage response and how they impact nuclear organization and transcription.

Nuclear myosins - roles for molecular transporters and anchors.

The myosin family of molecular motors are well-characterised cytoskeletal proteins. However, myosins are also present in the nucleus, where they have been shown to have roles in transcription, DNA repair and viral infections. Despite their involvement in these fundamental cellular processes, our understanding of these functions and their regulation remains limited. Recently, research on nuclear myosins has been gathering pace, and this Review will evaluate the current state of the field. Here, we will focus on the variation in structure of nuclear myosins, their nuclear import and their roles within transcription, DNA damage, chromatin organisation and viral infections. We will also consider both the biochemical and biophysical properties and restraints that are placed on these multifunctional motors, and how they link to their cytoplasmic counterparts. By highlighting these properties and processes, we show just how integral nuclear myosins are for cellular survival.