Biology subjects

Pu, S.

Publications and source records attributed to Pu, S..

3 recordsLinked to original sources

bioRxiv2020

Structural and functional characterization of the Spo11 core complex

Spo11, which makes DNA double-strand breaks (DSBs) essential for meiotic recombination, is poorly understood mechanistically because it has been recalcitrant to biochemical study. Here, we provide a molecular analysis of S. cerevisiae Spo11 purified with partners Rec102, Rec104 and Ski8. Rec102 and Rec104 jointly resemble the B subunit of archaeal Topoisomerase VI, with Rec104 similar to a GHKL domain but without conserved ATPase motifs. Unexpectedly, the Spo11 complex is monomeric (1:1:1:1 stoichiometry), indicating that dimerization may control DSB formation. Reconstitution of DNA binding reveals topoisomerase-like preferences for duplex-duplex junctions and bent DNA. Spo11 also binds noncovalently but with high affinity to DNA ends mimicking cleavage products, suggesting a mechanism to cap DSB ends. Mutations that reduce DNA binding in vitro attenuate DSB formation, alter DSB processing, and reshape the DSB landscape in vivo. Our data reveal structural and functional similarities between the Spo11 core complex and Topo VI, but also highlight differences reflecting their distinct biological roles.

molecular biology↗

bioRxiv2020

DNA-dependent macromolecular condensation drives self-assembly of the meiotic DNA break machinery

Formation of meiotic DNA double-strand breaks (DSBs) by Spo11 is tightly regulated and tied to chromosome structure, but the higher-order assemblies that execute and control DNA breakage are poorly understood. We address this question through molecular characterization of Saccharomyces cerevisiae RMM proteins (Rec114, Mei4 and Mer2)--essential, conserved components of the DSB machinery. Each subcomplex of Rec114-Mei4 (2:1 heterotrimer) or Mer2 (homotetrameric coiled coil) is monodisperse in solution, but they independently condense with DNA into dynamic, reversible nucleoprotein clusters that share properties with phase-separated systems. Multivalent interactions drive condensation, which correlates with DSB formation in vivo. Condensates fuse into mixed Rec114-Mei4-Mer2 clusters that further recruit Spo11 complexes. Our data show how the DSB machinery self-assembles on chromosome axes to create centers of DSB activity. We propose that multilayered control of Spo11 arises from recruitment of regulatory components and modulation of biophysical properties of the condensates.

molecular biology↗

bioRxiv2019

Regulation of transcription termination by FUS and TDP-43

The carboxy-terminal domain (CTD) of the RNA polymerase II (RNAPII) subunit POLR2A is a platform for modifications specifying the recruitment of factors that regulate transcription, mRNA processing, and chromatin remodelling. We previously found that symmetrical dimethylation (me2s) of a CTD Arginine residue (R1810 in human) causes recruitment of the Tudor domain of SMN, which interacts with Senataxin. SMN is mutated in spinal muscular atrophy (SMA), and Senataxin is sometimes mutated in Amyotrophic Lateral Sclerosis (ALS). R1810me2s and SMN, like Senataxin, are important for resolving R-loops (DNA:RNA hybrids) at transcription terminators. FUS and TDP-43 (TARDBP) are DNA/RNA binding proteins that are sometimes mutated in ALS and FTD (Frontotemporal dementia). Here we show that TDP-43 and, to some extent, FUS are recruited by the R1810me2s-SMN pathway. Defects in FUS and TDP-43 recruitment influence RNAPII termination and R-loop accumulation, leading to elevated DNA damage at terminators that may contribute to neurodegenerative disorders like ALS and FTD.

molecular biology↗