Cryo-EM structure of the Smc5/6 holo-complex

Abstract

The Smc5/6 complex plays an essential role in the resolution of recombination intermediates formed during mitosis or meiosis, or as a result of the cellular response to replication stress. It also functions as a restriction factor preventing viral replication. Here, we report the cryogenic EM (cryo-EM) structure of the six-subunit budding yeast Smc5/6 holo-complex, reconstituted from recombinant proteins expressed in insect cells – providing both an architectural overview of the entire complex and an understanding of how the Nse1/3/4 subcomplex binds to the hetero-dimeric SMC protein core. In addition, we demonstrate that a region within the head domain of Smc5, equivalent to the ‘W-loop’ of Smc4 or ‘F-loop’ of Smc1, mediates an important interaction with Nse1. Notably, mutations that alter the surface-charge profile of the region of Nse1 which accepts the Smc5-loop, lead to a slow-growth phenotype and a global reduction in the chromatin-associated fraction of the Smc5/6 complex, as judged by single molecule localisation microscopy experiments in live yeast. Moreover, when taken together, our data indicates functional equivalence between the structurally unrelated KITE and HAWK accessory subunits associated with SMC complexes.

DNA segment capture by Smc5/6 holocomplexes

“…All of them cause lethality when removed by gene disruption 10 . Smc5/6 and Nse4 together form the conserved ATPase core with an elongated shape that is characteristic for all SMC complexes [17][18][19][20][21][22] . Smc5 and Smc6 under protein-denaturing conditions (Fig.…”

The SAGA histone acetyltransferase module targets SMC5/6 to specific genes

“…Interestingly, the SMC5/6 subunits (Nse2, Nse3 and Smc5 arm) are aligned at the surface of the rod-shaped complex for binding to the SAGA complex (Fig 2 B and Additional file 1 : Fig. S4B; [ 15 ]). In contrast, the Nse3 subunit is mostly buried inside the ring-shaped complex (Additional file 1 : Fig.…”

“…The SAGA histone acetyltransferase (HAT; red) module binds several SMC5/6 subunits (top panel; Nse2: black, Smc5 arm: blue, and Nse3: green; model based on Ref. [ 15 ]). SAGA complex (violet; model based on Ref.…”

“…The Smc subunits are primarily built of head ATPase domains, long anti-parallel coiled-coil arms and hinges [ 11 – 13 ]. Two Smc molecules form stable dimers via their hinge domains, and without ATP, their arms align into rod-like structures [ 14 , 15 ]. The binding of ATP molecules to the ATPase head domains promotes the formation of large annular structures [ 16 , 17 ].…”

SAGA histone acetyltransferase module facilitates chromatin accessibility to SMC5/6

“…The Smc subunits are primarily built of head ATPase domains, long anti-parallel coiled-coil arms, and hinges [10–12]. Two Smc molecules form stable dimers via their hinge domains, and without ATP, their arms align into rod-like structures [13, 14]. The binding of ATP molecules to the ATPase head domains promotes the formation of large annular structures [15, 16].…”