Cancer-Associated Fibroblast Functions as a Road-Block in Cancer Therapy

The journey of a normal resident fibroblast belonging to the tumor microenvironment (TME) from being a tumor pacifier to a tumor patron is fascinating. We introduce cancer-associated fibroblast (CAF) as a crucial component of the TME. Activated-CAF partners with tumor cells and all components of TME in an established solid tumor. We briefly overview the origin, activation, markers, and overall functions of CAF with a particular reference to how different functions of CAF in an established tumor are functionally connected to the development of resistance to cancer therapy in solid tumors. We interrogate the role of CAF in mediating resistance to different modes of therapies. Functional diversity of CAF in orchestrating treatment resistance in solid tumors portrays CAF as a common orchestrator of treatment resistance; a roadblock in cancer therapy.

Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.

Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named “Rad53-dependent origins”. The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription.Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

Developmental differences in genome replication program and origin activation

To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.

Low replication stress leads to specific replication timing advances associated to chromatin remodelling in cancer cells

ABSTRACTDNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress.

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and −6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

Caught in the act: structural dynamics of replication origin activation and fork progression

This review discusses recent advances in single-particle cryo-EM and single-molecule approaches used to visualise eukaryotic DNA replication reactions reconstituted in vitro. We comment on the new challenges facing structural biologists, as they turn to describing the dynamic cascade of events that lead to replication origin activation and fork progression.

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

ABSTRACTEukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal tail of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, but requires Rad53 activation by trans-autophosphorylation, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.