Structural basis for plasmid restriction by SMC JET nuclease.

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.

SMC-based immunity against extrachromosomal DNA elements.

SMC and SMC-like complexes promote chromosome folding and genome maintenance in all domains of life. Recently, they were also recognized as factors in cellular immunity against foreign DNA. In bacteria and archaea, Wadjet and Lamassu are anti-plasmid/phage defence systems, while Smc5/6 and Rad50 complexes play a role in anti-viral immunity in humans. This raises an intriguing paradox - how can the same, or closely related, complexes on one hand secure the integrity and maintenance of chromosomal DNA, while on the other recognize and restrict extrachromosomal DNA? In this minireview, we will briefly describe the latest understanding of each of these complexes in immunity including speculations on how principles of SMC(-like) function may explain how the systems recognize linear or circular forms of invading DNA.

DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage.

Structural maintenance of chromosome (SMC) complexes fold DNA by loop extrusion to support chromosome segregation and genome maintenance. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC complexes with roles in bacterial immunity against selfish DNA. Here, we show that JetABCD restricts circular plasmids with an upper size limit of about 100 kb, whereas a linear plasmid evades restriction. Purified JetABCD complexes cleave circular DNA molecules, regardless of the DNA helical topology; cleavage is DNA sequence nonspecific and depends on the SMC ATPase. A cryo-EM structure reveals a distinct JetABC dimer-of-dimers geometry, with the two SMC dimers facing in opposite direction-rather than the same as observed with MukBEF. We hypothesize that JetABCD is a DNA-shape-specific endonuclease and propose the "total extrusion model" for DNA cleavage exclusively when extrusion of an entire plasmid has been completed by a JetABCD complex. Total extrusion cannot be achieved on the larger chromosome, explaining how self-DNA may evade processing.

A joint-ParB interface promotes Smc DNA recruitment.

Chromosomes readily unlink and segregate to daughter cells during cell division, highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes promote DNA organization by loop extrusion. In most bacteria, chromosome folding initiates at dedicated start sites marked by the ParB/parS partition complexes. Whether SMC complexes recognize a specific DNA structure in the partition complex or a protein component is unclear. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate foreign chromosomes. Using chimeric proteins and chemical cross-linking, we find that ParB directly binds the Smc subunit. We map an interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex, presumably to initiate DNA loop extrusion.

Bridging-induced phase separation induced by cohesin SMC protein complexes.

Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo-base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.

Division of Labor between PCNA Loaders in DNA Replication and Sister Chromatid Cohesion Establishment.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.

Structural studies of RFCCtf18 reveal a novel chromatin recruitment role for Dcc1.

Replication factor C complexes load and unload processivity clamps from DNA and are involved in multiple DNA replication and repair pathways. The RFCCtf18 variant complex is required for activation of the intra-S-phase checkpoint at stalled replication forks and aids the establishment of sister chromatid cohesion. Unlike other RFC complexes, RFCCtf18 contains two non-Rfc subunits, Dcc1 and Ctf8. Here, we present the crystal structure of the Dcc1-Ctf8 heterodimer bound to the C-terminus of Ctf18. We find that the C-terminus of Dcc1 contains three-winged helix domains, which bind to both ssDNA and dsDNA We further show that these domains are required for full recruitment of the complex to chromatin, and correct activation of the replication checkpoint. These findings provide the first structural data on a eukaryotic seven-subunit clamp loader and define a new biochemical activity for Dcc1.