No abstract

Degradation of the histone H4 methyltransferase SET8, which regulates chromosome compaction and genomic integrity, is regulated by the CRL4(CDT2) ubiquitin ligase to facilitate DNA replication and repair.

Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies. A free web tool for TIDER data analysis is available at http://tide.nki.nl.

No abstract

DNA replication fork perturbation is a major challenge to the maintenance of genome integrity. It has been suggested that processing of stalled forks might involve fork regression, in which the fork reverses and the two nascent DNA strands anneal. Here, we show that FBH1 catalyzes regression of a model replication fork in vitro and promotes fork regression in vivo in response to replication perturbation. Cells respond to fork stalling by activating checkpoint responses requiring signaling through stress-activated protein kinases. Importantly, we show that FBH1, through its helicase activity, is required for early phosphorylation of ATM substrates such as CHK2 and CtIP as well as hyperphosphorylation of RPA. These phosphorylations occur prior to apparent DNA double-strand break formation. Furthermore, FBH1-dependent signaling promotes checkpoint control and preserves genome integrity. We propose a model whereby FBH1 promotes early checkpoint signaling by remodeling of stalled DNA replication forks.

CtIP-dependent DNA end resection, which was previously thought to be necessary for CHK1 kinase activation and subsequent DNA damage checkpoint induction, is in fact only required for sustained checkpoint signaling.

The molecular events occurring following the disruption of DNA replication forks are poorly characterized, despite extensive use of replication inhibitors such as hydroxyurea in the treatment of malignancies. Here, we identify a key role for the FBH1 helicase in mediating DNA double-strand break formation following replication inhibition. We show that FBH1-deficient cells are resistant to killing by hydroxyurea, and exhibit impaired activation of the pro-apoptotic factor p53, consistent with decreased DNA double-strand break formation. Similar findings were obtained in murine ES cells carrying disrupted alleles of Fbh1. We also show that FBH1 through its helicase activity co-operates with the MUS81 nuclease in promoting the endonucleolytic DNA cleavage following prolonged replication stress. Accordingly, MUS81 and EME1-depleted cells show increased resistance to the cytotoxic effects of replication stress. Our data suggest that FBH1 helicase activity is required to eliminate cells with excessive replication stress through the generation of MUS81-induced DNA doublestrand breaks.

Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations ingenomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/218156 doi: bioRxiv preprint first posted online Nov. 13, 2017; 2 The CRISPR system for genome editing has become one of the most popular techniques in molecular biology. CRISPR endonucleases such as Cas9 can cleave genomic DNA with high precision, and due to error-prone repair mechanisms this can result in small insertions or deletions (indels) [1][2][3] .Alternatively, precisely designed small nucleotide changes can be incorporated near the break site by providing a donor template 4,5 , such as a single-stranded oligodeoxynucleotide (ssODN) 4,6 . By homology-directed repair (HDR), the DNA of the donor template is exchanged with the genomic DNA, and thereby the desired mutations are introduced 7,8 . Such precise editing offers the possibility to create and study specific mutations, or to correct disease-causing nucleotide variants 5,9 .A current limitation of this template-directed strategy is that the efficacy is unpredictable and often low. Because error-prone non-templated repair pathways are active besides HDR, various indels are often introduced at the target site instead of the desired mutation. Moreover, a substantial fraction of the target sequence may remain unaltered. Thus, exposing a pool of cells to CRISPR and a donor template yields a complex mixture of cells with wild-type DNA, indels and the designed mutation, with unpredictable ratios [10][11][12] . A quick and easy assay to determine these ratios is of key importance, particularly if one wants to estimate how many cells are to be cloned from the pool in order to obtain at least one clonal line with the desired mutation.High throughput sequencing of DNA around the induced break site is a powerful tool to analyze the mutation spectrum 13 , but is also expensive and requires substantial computational analysis. The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/218156 doi: bioRxiv preprint first posted online Nov. 13, 2017; 3 the sgRNA is used as input to determine the expected break site. Indels are then quantified by computational decomposition of the mixture of sequences in the test sequence trace, using the control sequence trace for comparison 14 .TIDER takes a similar decomposition approach, but it requires one additional capillary sequencing trace. This "reference" trace is derived from a pure DNA sample that c...