USP11 deubiquitinates RAE1 and plays a key role in bipolar spindle formation.

Correct segregation of the mitotic chromosomes into daughter cells is a highly regulated process critical to safeguard genome stability. During M phase the spindle assembly checkpoint (SAC) ensures that all kinetochores are correctly attached before its inactivation allows progression into anaphase. Upon SAC inactivation, the anaphase promoting complex/cyclosome (APC/C) E3 ligase ubiquitinates and targets cyclin B and securin for proteasomal degradation. Here, we describe the identification of Ribonucleic Acid Export protein 1 (RAE1), a protein previously shown to be involved in SAC regulation and bipolar spindle formation, as a novel substrate of the deubiquitinating enzyme (DUB) Ubiquitin Specific Protease 11 (USP11). Lentiviral knock-down of USP11 or RAE1 in U2OS cells drastically reduces cell proliferation and increases multipolar spindle formation. We show that USP11 is associated with the mitotic spindle, does not regulate SAC inactivation, but controls ubiquitination of RAE1 at the mitotic spindle, hereby functionally modulating its interaction with Nuclear Mitotic Apparatus protein (NuMA).

Serine is a new target residue for endogenous ADP-ribosylation on histones.

ADP-ribosylation (ADPr) is a biologically and clinically important post-translational modification, but little is known about the amino acids it targets on cellular proteins. Here we present a proteomic approach for direct in vivo identification and quantification of ADPr sites on histones. We have identified 12 unique ADPr sites in human osteosarcoma cells and report serine ADPr as a new type of histone mark that responds to DNA damage.

The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin.

UNLABELLED: Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. IMPORTANCE: Cell division requires unwinding of millions of DNA base pairs to generate the template for RNA transcripts as well as chromosome replication. As both processes use the same template, frequent clashes are unavoidable. To minimize the impact of these clashes, transcription and replication in bacteria follow the same directionality, thereby avoiding head-on collisions. This codirectionality is maintained by a strict regulation of where replication is started. We have used Escherichia coli as a model to investigate cells in which the defined location of replication initiation is compromised. In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises. In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells.

Avoiding chromosome pathology when replication forks collide.

Chromosome duplication normally initiates through the assembly of replication fork complexes at defined origins. DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet through a replisome assembly mechanism normally associated with fork repair, replication restart and recombination, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realized when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit several origins to replicate each chromosome.

On the viability of Escherichia coli cells lacking DNA topoisomerase I.

BACKGROUND: Manipulations of the DNA double helix during replication, transcription and other nucleic acid processing cause a change of DNA topology, which results in torsional stress. This stress is relaxed by DNA topoisomerases, a class of enzymes present in all domains of life. Negatively supercoiled DNA is relaxed by type IA topoisomerases that are widespread in bacteria, archaea and eukaryotes. In Escherichia coli there is conflicting data about viability of ΔtopA cells lacking topoisomerase I. RESULTS: In this study we sought to clarify whether E. coli cells lacking topoisomerase I are viable by using a plasmid-based lethality assay that allowed us to investigate the phenotype of ΔtopA cells without the presence of any compensatory mutations. Our results show that cells lacking topoisomerase I show an extreme growth defect and cannot be cultured without the accumulation of compensatory mutations. This growth defect can be partially suppressed by overexpression of topoisomerase III, the other type IA topoisomerase in E. coli, suggesting that the accumulation of torsional stress is, at least partially, responsible for the lethality of ΔtopA cells. The absence of RNase HI strongly exacerbates the phenotype of cells lacking topoisomerase I, which supports the idea that the processing of RNA:DNA hybrids is vitally important in ΔtopA cells. However, we did not observe suppression of the ΔtopA phenotype by increasing the level of R-loop processing enzymes, such as RNase HI or RecG. CONCLUSIONS: Our data show unambiguously that E. coli cells are not viable in the absence of DNA topoisomerase I without the presence of compensatory mutations. Furthermore, our data suggest that the accumulation of R-loops is not the primary reason for the severe growth defect of cells lacking topoisomerase I, which is in contrast to the current literature. Potential reasons for this discrepancy are discussed.