Structural basis for the E3 ligase activity enhancement of yeast Nse2 by SUMO-interacting motifs

Post-translational modification of proteins by ubiquitin and ubiquitin-like modifiers, such as SUMO, are key events in protein homeostasis or DNA damage response. Smc5/6 is a nuclear multi-subunit complex that participates in the recombinational DNA repair processes and is required in the maintenance of chromosome integrity. Nse2 is a subunit of the Smc5/6 complex that possesses SUMO E3 ligase activity by the presence of a SP-RING domain that activates the E2~SUMO thioester for discharge on the substrate. Here we present the crystal structure of the SUMO E3 ligase Nse2 in complex with an E2-SUMO thioester mimetic. In addition to the interface between the SP-RING domain and the E2, the complex reveals how two SIM (SUMO-Interacting Motif) -like motifs in Nse2 are restructured upon binding the donor and E2-backside SUMO during the E3-dependent discharge reaction. Both SIM interfaces are essential in the activity of Nse2 and are required to cope with DNA damage.

Managing Single-Stranded DNA during Replication Stress in Fission Yeast

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

Managing Single-Stranded DNA during Replication Stress in Fission Yeast

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

Natural variation in plant telomere length is associated with flowering time

Telomeres are highly repetitive DNA sequences found at the ends of chromosomes that protect the chromosomes from deterioration during cell division. Here, using whole genome re-sequencing and terminal restriction fragment assays, we found substantial natural intraspecific variation in telomere length in Arabidopsis thaliana, rice (Oryza sativa), and maize (Zea mays). Genome-wide association study (GWAS) mapping in A. thaliana identified 13 regions with GWAS-significant associations underlying telomere length variation, including a region that harbors the telomerase reverse transcriptase (TERT) gene. Population genomic analysis provided evidence for a selective sweep at the TERT region associated with longer telomeres. We found that telomere length is negatively correlated with flowering time variation not only in A. thaliana, but also in maize and rice, indicating a link between life history traits and chromosome integrity. Our results point to several possible reasons for this correlation, including the possibility that longer telomeres may be more adaptive in plants that have faster developmental rates (and therefore flower earlier). Our work suggests that chromosomal structure itself might be an adaptive trait associated with plant life history strategies.

A bacterial prophage small peptide counteracts DnaA activities in B. subtilis

Bacteriophages are able to hijack host essential machineries to benefit their fitness and assemble their own progeny. Phage proteins targeting major bacterial pathways can be powerful tools to understand cell functions and have possible applications in human health and industry. Bacterial genomes also harbor cryptic prophages carrying genes that may contribute to their host fitness and properties. The cryptic prophages are mostly transcriptionally silent and most of the functions they encode are not annotated. In B. subtilis, the 48 kb-long skin element is a prophage carrying the yqaF-yqaN operon, which is tightly regulated by the Xre-like repressor sknR. The small yqaH gene potentially encodes the protein YqaH in absence of SknR. It was previously reported that YqaH interacts with the replication initiator DnaA in yeast two-hybrid assay and its expression in B. subtilis causes defects in the chromosomal cycle. In this study, we report that, in addition to DnaA, YqaH interacts with Spo0A, a master regulator of sporulation. To decipher yqaH mode of action, we used the yeast two-hybrid to isolate single mutations in yqaH that separate interactions with DnaA and Spo0A. We isolated mutations that caused loss-of-interaction (LOI) with DnaA but not Spo0A. However, all mutations disrupting the interaction with Spo0A were also DnaA-LOI functions, suggesting that these functions could not be separated. We found that expression YqaH carrying DnaA-LOI mutations affects both chromosome integrity and DnaA-mediated transcription, leading to growth inhibition as well as preventing bacterial development such as sporulation and biofilm formation. These results show that YqaH acts as an antimicrobial peptide in B. subtilis and pave the way for the structural design of mutants with improved antibacterial action.

LINC00973 Induces Proliferation Arrest of Drug-Treated Cancer Cells by Preventing p21 Degradation

Overcoming drug resistance of cancer cells is the major challenge in molecular oncology. Here, we demonstrate that long non-coding RNA LINC00973 is up-regulated in normal and cancer cells of different origins upon treatment with different chemotherapeutics. Bioinformatics analysis shows that this is a consequence of DNA damage response pathway activation or mitotic arrest. Knockdown of LINC0973 decreases p21 levels, activates cellular proliferation of cancer cells, and suppresses apoptosis of drug-treated cells. We have found that LINC00973 strongly increases p21 protein content, possibly by blocking its degradation. Besides, we have found that ectopic over-expression of LINC00973 inhibits formation of the pro-survival p53-Ser15-P isoform, which preserves chromosome integrity. These results might open a new approach to the development of more efficient anti-cancer drugs.

Chromosome Integrity is Required for the Initiation of Meiotic Sex Chromosome Inactivation in Caenorhabditis elegans

During meiosis of heterogametic cells, such as XY meiocytes, sex chromosomes of many species undergo transcriptional silencing known as meiotic sex chromosome inactivation (MSCI). Silencing also occurs in aberrantly unsynapsed autosomal chromatin. The silencing of unsynapsed chromatin, is assumed to be the underline mechanism for MSCI. Initiation of MSCI is disrupted in meiocytes with sex chromosome-autosome translocations. Whether this is due to aberrant synapsis or the lack of sex chromosome integrity has never been determined. To address this, we used CRISPR to engineer Caenorhabditis elegans stable strains with broken X chromosomes that didn’t undergo translocations with autosomes. In early meiotic nuclei of these mutants, the X fragments lack silent chromatin modifications and instead the fragments are enriched with transcribing chromatin modifications. Moreover, the level of active RNA polymerase II staining on the X fragments in mutant nuclei is similar to that on autosomes, indicating active transcription on the X. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, X fragments that did not synapse were robustly stained with RNA polymerase II and gene expression levels were high throughout the broken X. Therefore, lack of synapsis does not trigger MSCI if sex chromosome integrity is lost. Moreover, our results suggest that a unique character of the chromatin of sex chromosomes underlies their lack of meiotic silencing due to both unsynapsed chromatin and sex chromosome mechanisms when their integrity is lost.

Telomere lengths in plants are correlated with flowering time variation

Telomeres are highly repetitive tandemly repeating DNA sequences found at chromosomal ends that protect chromosomes from deterioration during cell division. Using whole genome re-sequencing data, we found substantial natural intraspecific variation in telomere lengths in Arabidopsis thaliana, Oryza sativa (rice) and Zea mays (maize). Genome-wide association mapping in A. thaliana identifies a region that includes the telomerase reverse transcriptase (TERT) gene as underlying telomere length variation. TERT appears to exist in two haplotype groups (L and S), of which the L haplogroup allele shows evidence of a selective sweep in Arabidopsis. We find that telomere length is negatively correlated with flowering time variation not only in A. thaliana, but also in maize and rice, indicating a link between life history traits and chromosome integrity. We suggest that longer telomeres may be more adaptive in plants that have faster developmental rates (and therefore flower earlier), and that chromosomal structure itself is an adaptive trait associated with plant life history strategies.