Signatures of TOP1 transcription-associated mutagenesis in cancer and germline.

The mutational landscape is shaped by many processes. Genic regions are vulnerable to mutation but are preferentially protected by transcription-coupled repair1. In microorganisms, transcription has been demonstrated to be mutagenic2,3; however, the impact of transcription-associated mutagenesis remains to be established in higher eukaryotes4. Here we show that ID4-a cancer insertion-deletion (indel) mutation signature of unknown aetiology5 characterized by short (2 to 5 base pair) deletions -is due to a transcription-associated mutagenesis process. We demonstrate that defective ribonucleotide excision repair in mammals is associated with the ID4 signature, with mutations occurring at a TNT sequence motif, implicating topoisomerase 1 (TOP1) activity at sites of genome-embedded ribonucleotides as a mechanistic basis. Such TOP1-mediated deletions occur somatically in cancer, and the ID-TOP1 signature is also found in physiological settings, contributing to genic de novo indel mutations in the germline. Thus, although topoisomerases protect against genome instability by relieving topological stress6, their activity may also be an important source of mutations in the human genome.

An ERK5-KLF2 signalling module regulates early embryonic gene expression and telomere rejuvenation in stem cells.

The ERK5 MAP kinase signalling pathway drives transcription of naïve pluripotency genes in mouse Embryonic Stem Cells (mESCs). However, how ERK5 impacts on other aspects of mESC biology has not been investigated. Here, we employ quantitative proteomic profiling to identify proteins whose expression is regulated by the ERK5 pathway in mESCs. This reveals a function for ERK5 signalling in regulating dynamically expressed early embryonic 2-cell stage (2C) genes including the mESC rejuvenation factor ZSCAN4. ERK5 signalling and ZSCAN4 induction in mESCs increases telomere length, a key rejuvenative process required for prolonged culture. Mechanistically, ERK5 promotes ZSCAN4 and 2C gene expression via transcription of the KLF2 pluripotency transcription factor. Surprisingly, ERK5 also directly phosphorylates KLF2 to drive ubiquitin-dependent degradation, encoding negative feedback regulation of 2C gene expression. In summary, our data identify a regulatory module whereby ERK5 kinase and transcriptional activities bi-directionally control KLF2 levels to pattern 2C gene transcription and a key mESC rejuvenation process.

Structural and functional analysis of the GABARAP interaction motif (GIM).

Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X1-X2-[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a G ABARAP I nteraction M otif (GIM) sequence ([W/F]-[V/I]-X2-V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed 11-fold more specific for GABARAP than LC3B. Selective mutation of the X1 and X2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.

FAM111A regulates replication origin activation and cell fitness.

FAM111A is a replisome-associated protein and dominant mutations within its trypsin-like peptidase domain are linked to severe human developmental syndrome, the Kenny-Caffey syndrome. However, FAM111A functions remain unclear. Here, we show that FAM111A facilitates efficient activation of DNA replication origins. Upon hydroxyurea treatment, FAM111A-depleted cells exhibit reduced single-stranded DNA formation and a better survival rate. Unrestrained expression of FAM111A WT and patient mutants causes accumulation of DNA damage and cell death, only when the peptidase domain remains intact. Unrestrained expression of FAM111A WT also causes increased single-stranded DNA formation that relies on S phase entry, FAM111A peptidase activity but not its binding to proliferating cell nuclear antigen. Altogether, these data unveil how FAM111A promotes DNA replication under normal conditions and becomes harmful in a disease context.

Proteomic profiling reveals distinct phases to the restoration of chromatin following DNA replication.

Chromatin organization must be maintained during cell proliferation to preserve cellular identity and genome integrity. However, DNA replication results in transient displacement of DNA-bound proteins, and it is unclear how they regain access to newly replicated DNA. Using quantitative proteomics coupled to Nascent Chromatin Capture or isolation of Proteins on Nascent DNA, we provide time-resolved binding kinetics for thousands of proteins behind replisomes within euchromatin and heterochromatin in human cells. This shows that most proteins regain access within minutes to newly replicated DNA. In contrast, 25% of the identified proteins do not, and this delay cannot be inferred from their known function or nuclear abundance. Instead, chromatin organization and G1 phase entry affect their reassociation. Finally, DNA replication not only disrupts but also promotes recruitment of transcription factors and chromatin remodelers, providing a significant advance in understanding how DNA replication could contribute to programmed changes of cell memory.

Absence of DEATH kinesin is fatal for Leishmania mexicana amastigotes.

Kinesins are motor proteins present in organisms from protists to mammals playing important roles in cell division, intracellular organisation and flagellum formation and maintenance. Leishmania mexicana is a protozoan parasite of the order Kinetoplastida causing human cutaneous leishmaniasis. Kinetoplastida genome sequence analyses revealed a large number of kinesins showing sequence and structure homology to eukaryotic kinesins. Here, we investigate the L. mexicana kinesin LmxKIN29 (LmxM.29.0350), also called DEATH kinesin. The activated MAP kinase LmxMPK3, a kinase affecting flagellum length in Leishmania, is able to phosphorylate recombinant full length LmxKIN29 at serine 554. Insect promastigote LmxKIN29 Leishmania null mutants showed no obvious phenotype. However, in mouse infection experiments, the null mutants were unable to cause the disease, whereas LmxKIN29 add-backs and single allele knockouts caused footpad lesions. Localisation using promastigotes expressing GFP-tagged LmxKIN29 revealed that the kinesin is predominantly found in between the nucleus and the flagellar pocket, while in dividing cells the GFP-fusion protein was found at the anterior and posterior ends of the cells indicating a role in cytokinesis. The inability to cause lesions in infected animals and the amino acid sequence divergence from mammalian kinesins suggests that LmxKIN29 is a potential drug target against leishmaniasis.