Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Summary Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large Lamina Associated Domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, consistency of NL contacts is inversely linked to gene activity in single cells, and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single cell chromatin organization.

Highlights d Profiled spatiotemporal gene expression patterns in human cardiogenesis d Mapped cell-type distribution and spatial organization in the human embryonic heart d Thoroughly analyzed roles of diverse cell types in cardiac development d A publicly available web resource of the human embryonic heart

We present a genome-wide method to map DNA double-strand breaks (DSBs) at nucleotide resolution by direct in situ breaks labeling, enrichment on streptavidin, and next-generation sequencing (BLESS). We comprehensively validated and tested BLESS using different human and mouse cells, DSBs-inducing agents, and sequencing platforms. BLESS was able to detect telomere ends, Sce endonuclease-induced DSBs, and complex genome-wide DSBs landscapes. As a proof of principle, we characterized the genomic landscape of sensitivity to replication stress in human cells, and identified over two thousand non-uniformly distributed aphidicolin-sensitive regions (ASRs) overrepresented in genes and enriched in satellite repeats. ASRs were also enriched in regions rearranged in human cancers, with many cancer-associated genes exhibiting high sensitivity to replication stress. Our method is suitable for genome-wide mapping of DSBs in various cells and experimental conditions with a specificity and resolution unachievable by current techniques.

Single-cell genomics and single-cell transcriptomics have recently emerged as powerful tools to study the biology of single cells at a genome-wide scale. However, a major challenge is to quantify both genomic DNA and mRNA from the same cell, which would allow direct comparison of genomic variation and transcriptome heterogeneity. Here we describe a method that allows the sequencing of genomic DNA and mRNA from the same cell without physical separation of the nucleic acids prior to amplification. We show that such an integrated strategy achieves efficiency similar to methods that sequence either genomic DNA or mRNA from single cells. We use this method to correlate DNA copy number variation to transcriptome variability among individual cells. Finally, we show that genes that display more cell-to-cell variability in transcript numbers are generally associated with reduced copy number loci and vice-versa, implying that copy number variations could potentially drive variability in gene expression between single cells.

Considerable progress in sequencing technologies makes it now possible to study the genomic and transcriptomic landscape of single cells. However, to better understand the complexity of multicellular organisms, we must devise ways to perform high-throughput measurements while preserving spatial information about the tissue context or subcellular localization of analysed nucleic acids. In this Innovation article, we summarize pioneering technologies that enable spatially resolved transcriptomics and discuss how these methods have the potential to extend beyond transcriptomics to encompass spatially resolved genomics, proteomics and possibly other omic disciplines.

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.Both prokaryotic and eukaryotic cells are endowed with multiple specialized DNA polymerases that are devoid of 3Ј35Ј proofreading exonuclease activity and replicate undamaged DNA in vitro with low fidelity and weak processivity (5). These specialized enzymes support DNA synthesis past a spectrum of template strand base damage by a process called translesion DNA synthesis (TLS), a mode of DNA damage tolerance that is fundamental to the survival of cells that suffer arrested DNA replication associated with damage to DNA.REV1 protein (which is confined to the eukaryotic kingdom) is a member of the Y family of DNA polymerases (14, 21). However, in vitro, the nucleotidyl transferase activity of REV1 is limited to the incorporation of just one or two dCMP moieties in a template-directed manner, regardless of the template nucleotide composition (19,30). This catalytic activity supports TLS past sites of base loss in vitro (19) and conceivably subserves this function in vivo. However, REV1 protein is also required for mutagenesis in both yeast and mammalian cells exposed to DNA-damaging agents that are not associated with the generation of sites of base loss, such as UV radiation (14). Remarkably, the dCMP transferase activity is dispensable for this function (1,14,18). Indeed, inactivation of the dCMP transferase activity in yeast does not result in defects in DNA damage-associated mutagenesis (9). Furthermore, a yeast mutant strain with a missense mutation in the N-terminal BRCT domain of REV1 retains dCMP transferase activity in vitro, even though it is deficient in TLS past sites of base loss and photoproducts (18).Several laboratories have demonstrated that the C-terminal ϳ100 amino acids of both mouse REV1 (mREV1) and human REV1 proteins can interact with multiple specialized DNA polymerases implicated in TLS (6,17,20,27). Additionally, different specialized DNA polymerases can compete with one another for binding to REV1 in vitro (6). Collectively, these observations suggest a presently unknown role(s) for REV1 in TLS that is unrelated to its dCMP transferase function.REV1 protein colocalizes with proliferating cell nuclear antigen (PCNA) in replication factories (27) and binds to other members of the Y family of DNA polymerases, to which it belongs, in...