Xiaolan Zhao

Mutations of the human SMC5/6 genome maintenance complex can lead to several genome instability syndromes and are associated with tumorigenesis. However, the molecular consequences of SMC5/6 deficiencies in human cells are only partly known, preventing a better understanding of disease-causing mechanisms. To address this issue, we employed the dTAG technology to induce rapid loss of SMC5 or SMC6 in a panel of human cell lines. While SMC5/6 depletion confers gradual cessation of cell proliferation in all tested cell lines, distinct molecular consequences are seen in p53-deficient verses -proficient cells. In p53-deficient HEK293A and HeLa cells or p53 knockout RPE1 cells, SMC5/6 depletion reveals a mitotic phenotype of increases in multipolar spindle and abnormal chromosome segregation. In contrast, in p53-proficient RPE1 and HCT116 cells, SMC5/6 loss favors G1 accumulation and thereby masking mitotic defects. The mitotic phenotype and cell proliferation defect caused by SMC5/6 loss is exacerbated by knockout of the BLM helicase, a tumor suppressor mutated in Bloom syndrome patients. In comparison, SMC5/6 did not show genetic interaction in cell proliferation assays with the MUS81 endo nuclease, the FANCM DNA helicase, or the PrimPol polymerase. Collectively, our data reveal that SMC5/6 is required to prevent multipolar spindle and associated mitotic defects in conjunction with BLM, thus providing new perspectives in understanding etiologies of diseases caused by their deficiencies.

The SUMO E3 enzymes control the efficiency and specificity of protein SUMOylation, providing regulatory means for many cellular processes. While most SUMO E3s fulfill their roles as single proteins, the conserved Nse2 E3 is an obligatory subunit of the genome-protecting complex Smc5/6. How the Smc5/6 complex functions in SUMOylation and the roles of its non-SUMO E3 subunits in this process remain to be elucidated. Here we examine the budding yeast Smc5/6 in SUMOylation reactions and in cellular SUMOylation assays. Biochemical data show that DNA stimulates Smc5/6’s E3 activity by fostering enzyme and substrate proximity. Mutational analyses reveal that four non-SUMO E3 subunits utilize their DNA-binding abilities to support this stimulation. Moreover, ATP binding by SMC subunits favors SUMOylation by enhancing Smc5/6 association with DNA and chromatin and by enabling conformational changes. Our findings thus provide evidence for a specialized DNA- and ATP-stimulated composite SUMO E3 complex that uses inter-subunit collaboration to achieve efficient SUMOylation in genome regulation.

Structural Maintenance of Chromosomes (SMC) complexes play critical roles in genome maintenance. While canonical SMCs, such as cohesin and condensin, organize the genome into 3D structures, Smc5/6 directly promotes genome replication and repair. Insights into Smc5/6 function have emerged from recent fluorescence-based single-molecule studies showing its binding to structures resembling replication and repair intermediates. To provide a higher-resolution view of this facet of Smc5/6′s function, we employed atomic force microscopy (AFM) to visualize the budding yeast Smc5/6 at nanometer-scale resolution and investigate how it interacts with short ssDNA gaps that frequently form during genome maintenance. We report that the free or DNA-bound Smc5/6 adopts a ‘bead-on-stick’ shape, with its head-NSE module separated from its hinge domain by a non-bending arm region. Smc5/6 predominantly uses its head-NSE module to engage with dsDNA, and mutating DNA-binding residues within this structural module shifts the complex toward a hinge-mediated DNA-binding mode. Importantly, Smc5/6 shows an intrinsic association preference for ssDNA gap regions and ss-dsDNA junction sites over the flanking dsDNA regions, predominantly through the head-NSE module. AFM visualization of Smc5/6 and its structural elements in preferential gap and junction DNA association provides a framework for our continued understanding of Smc5/6′s roles in genome maintenance.

Palindromic sequences are a potent source of genomic instability that can lead to cancer and hereditary diseases in humans. Molecular evidence shows that palindrome instability results from the formation of secondary structures, such as hairpins and cruciforms, which are cleaved by structure-specific nucleases. However, the mechanisms underlying cruciform formation and cleavage at palindromic sequences in eukaryotic cells remain incompletely understood. Here, we describe a pathway for secondary structure formation and chromosomal breakage at palindromes involving DNA helicase Mph1, Rad51 recombinase, Rad54 ATPase DNA strand remodeler, Rtt107 scaffold protein, and the multifunctional Smc5/6 complex. Deletion or mutation of any of these components results in a similar reduction in double-strand breaks at an Alu palindrome and a significant decrease in chromosomal rearrangements. We propose that Mph1, Rad51, and Rad54 work together at stalled replication forks to generate cruciform structures via fork remodeling, while Smc5/6 and Rtt107 mark the cruciforms, indicating an appropriate substrate for nuclease cleavage. As members of this pathway are conserved in humans, the uncovered mechanisms may underlie genomic instability at palindrome sites involved in disease etiology.

Smc5/6 is an essential genome maintenance complex that interacts with double-stranded (ds) DNA, single-stranded (ss) DNA, and ss-dsDNA junctions. DNA association underlies Smc5/6’s functions in managing intermediates generated during genome replication and repair. However, the mechanisms of this activity are not fully understood. Here, we report a single-molecule study examining Smc5/6 association with a dsDNA substrate containing a ssDNA gap with defined 3’ and 5’ junctions. We found that Smc5/6 associates with both 3’ and 5’ junctions but prefers the 3’ junction in the presence of the ssDNA-binding complex RPA. Further, Smc5/6’s junction association frequency and dwell time are regulated by two non-SMC subcomplexes and DNA binding residues of Smc6. Moreover, Smc5/6 prefers binding to junction sites free of the sliding clamp PCNA over those occupied with it. These results suggest that Smc5/6 utilizes its multiple structural modules to associate with junction sites in coordination with other genome maintenance factors.

The Structural Maintenance of Chromosomes (SMC) 5/6 complex is highly conserved and essential for mammalian development. SMC5/6 dysfunction in cells, model organisms, and patients with germline variants results in genome instability, though the exact function of the complex in genome maintenance remains enigmatic. Despite the importance of SMC5/6 in maintaining genome stability, the prevalence and consequences of somatic inactivation of SMC5/6 in cancer is understudied. Here we report a pan-cancer analysis of SMC5/6 dysfunction in cancer across three large databases. We identified thousands of tumors across all tissue types with copy number alteration and/or small variants in SMC5/6 genes. We found that deleterious variants in SMC5/6, but not copy number alterations, are associated with elevated tumor mutational burden (TMB). Mutagenesis in tumors with SMC5/6 variants was caused by polymerase epsilon (POLE) dysfunction and mismatch repair deficiency (MMRd). Patients in which SMC5/6 gene variants occur in tumor genomes exhibited improved survival relative to patients with non-altered SMC5/6 genes. This survival benefit was explained in part by a superior response to immunotherapy in a cohort of patients with colorectal cancer. Our findings demonstrate that dysfunction of SMC5/6 is predictive of elevated TMB and susceptibility to immunotherapy, indicating that SMC5/6 gene status should be considered in cancer diagnostic studies for prognostic implications and tailored therapeutics.

Many cancers use an alternative lengthening of telomeres (ALT) pathway for telomere maintenance. ALT telomeric DNA synthesis occurs in ALT-associated PML bodies (APBs). However, the mechanisms by which APBs form are not well understood. Here, we monitored the formation of APBs with time-lapse imaging employing CRISPR knock-in to track the promyelocytic leukemia (PML) protein at endogenous levels. We found APBs form via two pathways: telomeres recruit PML proteins to nucleate PML bodies de novo, or telomeres fuse with preformed PML bodies. Both nucleation and fusion of APBs require interactions between SUMO and SUMO interaction motifs (SIMs). Moreover, APB nucleation is associated with higher levels of SUMO and SUMO-mediated recruitment of DNA helicase BLM, resulting in more robust telomeric DNA synthesis. Finally, further boosting SUMO levels at telomeres enhances APB nucleation, BLM enrichment, and telomeric DNA synthesis. Thus, high SUMO levels at telomeres promote APB nucleation and stronger ALT activity.

SUMMARY: The ring-shaped sliding clamp PCNA enables DNA polymerases to perform processive DNA synthesis during replication and repair. The loading of PCNA onto DNA is catalyzed by the ATPase clamp loader RFC. Using a single-molecule platform to visualize the dynamic interplay between PCNA and RFC on DNA, we unexpectedly discovered that RFC continues to associate with PCNA after loading, contrary to the conventional view. Functionally, this clamp-loader/clamp complex is required for processive DNA synthesis by polymerase δ (Polδ), as the PCNA-Polδ assembly is inherently unstable. This architectural role of RFC is dependent on the BRCT domain of Rfc1, and mutation of its DNA-binding residues causes sensitivity to DNA damage in vivo. We further showed the FEN1 flap endonuclease can also stabilize the PCNA-Polδ interaction and mediate robust synthesis. Overall, our work revealed that, beyond their canonical enzymatic functions, PCNA-binding proteins harbor non-catalytic functions essential for DNA replication and genome maintenance.

Activation of the DNA damage checkpoint upon genotoxin treatment induces a multitude of cellular changes to cope with genome stress. After prolonged genotoxin treatment, the checkpoint can be downregulated to allow cell cycle and growth resumption. In yeast, downregulation of the DNA damage checkpoint requires the Srs2 DNA helicase, which removes the ssDNA binding complex replication protein A (RPA) and the associated Mec1 checkpoint kinase from DNA, thus dampening Mec1-mediated checkpoint. However, it is unclear whether the ‘anti-checkpoint’ role of Srs2 is temporally and spatially regulated to allow timely checkpoint termination while preventing superfluous RPA removal. Here we address this question by examining regulatory elements of Srs2, such as its phosphorylation, sumoylation, and protein-interaction sites. Our genetic analyses and checkpoint level assessment suggest that the RPA countering role of Srs2 is promoted by Srs2 binding to proliferating cell nuclear antigen (PCNA), which recruits Srs2 to a subset of ssDNA containing regions. RPA antagonism is further fostered by Srs2 sumoylation, which we found depends on the Srs2-PCNA interaction and Mec1, and peaks after Mec1 activity reaches maximal levels. These data support a model in which Srs2 recruitment to PCNA adjacent to ssDNA-RPA filaments, followed by Mec1-dependent sumoylation, modulates RPA-mediated checkpoint signaling, while Srs2 action is limited at ssDNA regions lacking proximal PCNA, thereby favoring RPA-mediated ssDNA protection and repair.