Structure and subunit topology of the INO80 chromatin remodeler and its nucleosome complex.

INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A.Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our analysis establishes a structural and functional framework for this family of large remodelers.

Mec1, INO80, and the PAF1 complex cooperate to limit transcription replication conflicts through RNAPII removal during replication stress.

Little is known about how cells ensure DNA replication in the face of RNA polymerase II (RNAPII)-mediated transcription, especially under conditions of replicative stress. Here we present genetic and proteomic analyses from budding yeast that uncover links between the DNA replication checkpoint sensor Mec1-Ddc2 (ATR-ATRIP), the chromatin remodeling complex INO80C (INO80 complex), and the transcription complex PAF1C (PAF1 complex). We found that a subset of chromatin-bound RNAPII is degraded in a manner dependent on Mec1, INO80, and PAF1 complexes in cells exposed to hydroxyurea (HU). On HU, Mec1 triggers the efficient removal of PAF1C and RNAPII from transcribed genes near early firing origins. Failure to evict RNAPII correlates inversely with recovery from replication stress: paf1Δ cells, like ino80 and mec1 mutants, fail to restart forks efficiently after stalling. Our data reveal unexpected synergies between INO80C, Mec1, and PAF1C in the maintenance of genome integrity and suggest a mechanism of RNAPII degradation that reduces transcription-replication fork collision.

The nuclear actin-containing Arp8 module is a linker DNA sensor driving INO80 chromatin remodeling.

Nuclear actin (N-actin) and actin-related proteins (Arps) are critical components of several chromatin modulating complexes, including the chromatin remodeler INO80, but their function is largely elusive. Here, we report the crystal structure of the 180-kDa Arp8 module of Saccharomyces cerevisiae INO80 and establish its role in recognition of extranucleosomal linker DNA. Arp8 engages N-actin in a manner distinct from that of other actin-fold proteins and thereby specifies recruitment of the Arp4-N-actin heterodimer to a segmented scaffold of the helicase-SANT-associated (HSA) domain of Ino80. The helical HSA domain spans over 120 Å and provides an extended binding platform for extranucleosomal entry DNA that is required for nucleosome sliding and genome-wide nucleosome positioning. Together with the recent cryo-electron microscopy structure of INO80Core-nucleosome complex, our findings suggest an allosteric mechanism by which INO80 senses 40-bp linker DNA to conduct highly processive chromatin remodeling.

Structural basis for dodecameric assembly states and conformational plasticity of the full-length AAA+ ATPases Rvb1 · Rvb2.

As building blocks of diverse macromolecular complexes, the AAA+ ATPases Rvb1 and Rvb2 are crucial for many cellular activities including cancer-related processes. Their oligomeric structure and function remain unclear. We report the crystal structures of full-length heteromeric Rvb1·Rvb2 complexes in distinct nucleotide binding states. Chaetomium thermophilum Rvb1·Rvb2 assemble into hexameric rings of alternating molecules and into stable dodecamers. Intriguingly, the characteristic oligonucleotide-binding (OB) fold domains (DIIs) of Rvb1 and Rvb2 occupy unequal places relative to the compact AAA+ core ring. While Rvb1's DII forms contacts between hexamers, Rvb2's DII is rotated 100° outward, occupying lateral positions. ATP was retained bound to Rvb1 but not Rvb2 throughout purification, suggesting nonconcerted ATPase activities and nucleotide binding. Significant conformational differences between nucleotide-free and ATP-/ADP-bound states in the crystal structures and in solution suggest that the functional role of Rvb1·Rvb2 is mediated by highly interconnected structural switches. Our structures provide an atomic framework for dodecameric states and Rvb1·Rvb2's conformational plasticity.