DNA Damage-inducible Nuclear Microtubules Mobilize Rad52 Liquid Droplets to Mediate Repair

Damaged DNA exhibits increased mobility to promote repair. In Saccharomyces cerevisiae, repair of DNA double-strand breaks (DSBs) is driven by their clustering into nucleoplasmic DNA repair centres or mobilization to the nuclear periphery. This apparently random mobility is paradoxically abrogated upon disruption of microtubules or kinesins, which typically mediate the directional movement of macromolecules. We herein resolve this paradox by characterizing novel DNA damage-inducible intranuclear microtubule filaments (DIMs) that mobilize damaged DNA and promote repair. DNA damage-induced relief of centromeric constraint triggers DIMs that subsequently capture and directionally mobilize DNA lesions. Repressing and hyper-inducing DIMs abrogates and hyper-activates DNA repair, respectively. Accounting for DIM dynamics, by measuring directional changes of DSBs across cell populations, reveals an increased non-linear directional behavior in nuclear space. Abrogation of DIM-dependent processes or repair-promoting factors decreases directional behavior. We further reveal that the DNA repair protein Rad52 forms liquid droplets at different sites of DNA damage. Forces generated by the mechanical motions of short DIMs subsequently promote the coalescence of Rad52 droplets into repair centres. Upon coalescence, the large Rad52 droplet concentrates tubulin to connect the repair centre droplet to elongated DIMs for kinesin motor-dependent targeting of DSBs to the nuclear periphery and repair. Thus, inducible nuclear microtubule filaments cooperate with repair protein liquid droplets to cluster and directionally mobilize damaged DNA for repair.Ph.D

Nucleolar RNA polymerase II drives ribosome biogenesis

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli . Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease