Vertebrate DNA damage tolerance requires the C-terminus but not BRCT or transferase domains of REV1

REV1 is central to the DNA damage response of eukaryotes through an as yet poorly understood role in translesion synthesis. REV1 is a member of the Y-type DNA polymerase family and is capable of in vitro deoxycytidyl transferase activity opposite a range of damaged bases. However, non-catalytic roles for REV1 have been suggested by the Saccharomyces cerevisiae rev1-1 mutant, which carries a point mutation in the N-terminal BRCT domain, and the recently demonstrated ability of the mammalian protein to interact with each of the other translesion polymerases via its extreme C-terminus. Here, we show that a region adjacent to this polymerase interacting domain mediates an interaction with PCNA. These C-terminal domains of REV1 are necessary, although not sufficient, for effective tolerance of DNA damage in the avian cell line DT40, while the BRCT domain and transferase activity are not directly required. Together these data provide strong support for REV1 playing an important non-catalytic role in coordinating translesion synthesis. Further, unlike in budding yeast, rad18 is not epistatic to rev1 for DNA damage tolerance suggesting that REV1 and RAD18 play largely independent roles in the control of vertebrate translesion synthesis

The KRAB Zinc Finger Protein Roma/Zfp157 Is a Critical Regulator of Cell-Cycle Progression and Genomic Stability.

Regulation of DNA replication and cell division is essential for tissue growth and maintenance of genomic integrity and is particularly important in tissues that undergo continuous regeneration such as mammary glands. We have previously shown that disruption of the KRAB-domain zinc finger protein Roma/Zfp157 results in hyperproliferation of mammary epithelial cells (MECs) during pregnancy. Here, we delineate the mechanism by which Roma engenders this phenotype. Ablation of Roma in MECs leads to unscheduled proliferation, replication stress, DNA damage, and genomic instability. Furthermore, mouse embryonic fibroblasts (MEFs) depleted for Roma exhibit downregulation of p21Cip1 and geminin and have accelerated replication fork velocities, which is accompanied by a high rate of mitotic errors and polyploidy. In contrast, overexpression of Roma in MECs halts cell-cycle progression, whereas siRNA-mediated p21Cip1 knockdown ameliorates, in part, this phenotype. Thus, Roma is an essential regulator of the cell cycle and is required to maintain genomic stability.This work was supported by a PhD studentship from A*STAR Singapore to T.L.F.H. and funding from the Medical Research Council to C.J.W. G.G. and J.E.S. are supported by an MRC core grant to LMB (U105178808).This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.celrep.2016.03.07

Visualisation of PCNA Monoubiquitination In Vivo by Single Pass Spectral Imaging FRET Microscopy

Monoubiquitination of the DNA sliding clamp, PCNA, plays a central role in the control of damage bypass during replication. By combining a widely-spaced FRET donor/acceptor pair (CFP and mRFP) with spectral imaging, we have developed a simple method for the visualisation of PCNA monoubiquitination in both fixed and live cells with a single imaging pass. We validate the method with genetic controls in the avian cell line DT40 and use it to examine the intracellular dynamics of PCNA ubiquitination following subnuclear UV irradiation. This general approach is likely to be of utility for live imaging of post-translational modifications of a wide range of substrates in vivo

The Werner's Syndrome protein collaborates with REV1 to promote replication fork progression on damaged DNA

AbstractDNA damage tolerance pathways facilitate the bypass of DNA lesions encountered during replication. These pathways can be mechanistically divided into recombinational damage avoidance and translesion synthesis, in which the lesion is directly bypassed by specialised DNA polymerases. We have recently shown distinct genetic dependencies for lesion bypass at and behind the replication fork in the avian cell line DT40, bypass at the fork requiring REV1 and bypass at post-replicative gaps requiring PCNA ubiquitination by RAD18. The WRN helicase/exonuclease, which is mutated in the progeroid and cancer predisposition disorder Werner's Syndrome, has previously been implicated in a RAD18-dependent DNA damage tolerance pathway. However, WRN has also been shown to be required to maintain normal replication fork progression on a damaged DNA template, a defect reminiscent of REV1-deficient cells. Here we use the avian cell line DT40 to demonstrate that WRN assists REV1-dependent translesion synthesis at the replication fork and that PCNA ubiquitination-dependent post-replicative lesion bypass provides an important backup mechanism for damage tolerance in the absence of WRN protein

The intersection of DNA replication with antisense 3' RNA processing in Arabidopsis FLC chromatin silencing.

Funder: Wellcome TrustHow noncoding transcription influences chromatin states is still unclear. The Arabidopsis floral repressor gene FLC is quantitatively regulated through an antisense-mediated chromatin silencing mechanism. The FLC antisense transcripts form a cotranscriptional R-loop that is dynamically resolved by RNA 3' processing factors (FCA and FY), and this is linked to chromatin silencing. Here, we investigate this silencing mechanism and show, using single-molecule DNA fiber analysis, that FCA and FY are required for unimpeded replication fork progression across the Arabidopsis genome. We then employ the chicken DT40 cell line system, developed to investigate sequence-dependent replication and chromatin inheritance, and find that FLC R-loop sequences have an orientation-dependent ability to stall replication forks. These data suggest a coordination between RNA 3' processing of antisense RNA and replication fork progression in the inheritance of chromatin silencing at FLC

PrimPol is required for replicative tolerance of G quadruplexes in vertebrate cells

G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments

DNA polymerase stalling at structured DNA constrains the expansion of short tandem repeats.

BACKGROUND: Short tandem repeats (STRs) contribute significantly to de novo mutagenesis, driving phenotypic diversity and genetic disease. Although highly diverse, their repetitive sequences induce DNA polymerase slippage and stalling, leading to length and sequence variation. However, current studies of DNA synthesis through STRs are restricted to a handful of selected sequences, limiting our broader understanding of their evolutionary behaviour and hampering the characterisation of the determinants of their abundance and stability in eukaryotic genomes. RESULTS: We perform a comprehensive analysis of DNA synthesis at all STR permutations and interrogate the impact of STR sequence and secondary structure on their genomic representation and mutability. To do this, we developed a high-throughput primer extension assay that allows monitoring of the kinetics and fidelity of DNA synthesis through 20,000 sequences comprising all STR permutations in different lengths. By combining these measurements with population-scale genomic data, we show that the response of a model replicative DNA polymerase to variously structured DNA is sufficient to predict the complex genomic behaviour of STRs, including abundance and mutational constraints. We demonstrate that DNA polymerase stalling at DNA structures induces error-prone DNA synthesis, which constrains STR expansion. CONCLUSIONS: Our data support a model in which STR length in eukaryotic genomes results from a balance between expansion due to polymerase slippage at repeated DNA sequences and point mutations caused by error-prone DNA synthesis at DNA structures

Y-family DNA polymerases and their role in tolerance of cellular DNA damage.

The past 15 years have seen an explosion in our understanding of how cells replicate damaged DNA and how this can lead to mutagenesis. The Y-family DNA polymerases lie at the heart of this process, which is commonly known as translesion synthesis. This family of polymerases has unique features that enable them to synthesize DNA past damaged bases. However, as they exhibit low fidelity when copying undamaged DNA, it is essential that they are only called into play when they are absolutely required. Several layers of regulation ensure that this is achieve

A p53-Dependent Checkpoint Induced upon DNA Damage Alters Cell Fate during hiPSC Differentiation.

The ability of human induced pluripotent stem cells (hiPSCs) to differentiate in vitro to each of the three germ layer lineages has made them an important model of early human development and a tool for tissue engineering. However, the factors that disturb the intricate transcriptional choreography of differentiation remain incompletely understood. Here, we uncover a critical time window during which DNA damage significantly reduces the efficiency and fidelity with which hiPSCs differentiate to definitive endoderm. DNA damage prevents the normal reduction of p53 levels as cells pass through the epithelial-to-mesenchymal transition, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53-deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a "differentiation checkpoint" in early endoderm differentiation that alters cell fate in response to DNA damage