Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1

Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities

C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast

Kinetochores are macromolecular protein assemblies at centromeres that mediate accurate chromosome segregation during cell division. The outer kinetochore KNL1SPC105, MIS12MTW1, and NDC80NDC80 complexes assemble the KMN network, which harbors the sites of microtubule binding and spindle assembly checkpoint signaling. The buildup of the KMN network that transmits microtubule pulling forces to budding yeast point centromeres is poorly understood. Here, we identify 225 inter-protein crosslinks by mass spectrometry on KMN complexes isolated from Saccharomyces cerevisiae that delineate the KMN subunit connectivity for outer kinetochore assembly. C-Terminal motifs of Nsl1 and Mtw1 recruit the SPC105 complex through Kre28, and both motifs aid tethering of the NDC80 complex by the previously reported Dsn1 C terminus. We show that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association

The COMA complex interacts with Cse4 and positions Sli15/Ipl1 at the budding yeast inner kinetochore

International audienceKinetochores are macromolecular protein complexes at centromeres that ensure accurate chromosome segregation by attaching chromosomes to spindle microtubules and integrating safeguard mechanisms. The inner kinetochore is assembled on CENP-A nucleosomes and has been implicated in establishing a kinetochore-associated pool of Aurora B kinase, a chromosomal passenger complex (CPC) subunit, which is essential for chromosome biorientation. By performing crosslink-guided in vitro reconstitution of budding yeast kinetochore complexes we showed that the Ame1/Okp1CENP-U/Q heterodimer, which forms the COMA complex with Ctf19/Mcm21CENP-P/O, selectively bound Cse4CENP-A nucleosomes through the Cse4 N-terminus. The Sli15/Ipl1INCENP/Aurora-B core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affects accurate chromosome segregation in a Sli15 wild-type background. Tethering Sli15 to Ame1/Okp1 rescued synthetic lethality upon Ctf19 depletion in a Sli15 centromere-targeting deficient mutant. This study shows molecular characteristics of the point-centromere inner kinetochore architecture and suggests a role for the Ctf19 C-terminus in mediating accurate chromosome segregation