Dynamic configurations of meiotic DNA-break hotspot determinant proteins

Appropriate DNA double-strand-break (DSB) and crossover distributions are required for proper meiotic chromosome segregation. Schizosaccharomyces pombe linear element proteins (LinEs) determine DSB hotspots; LinE-bound hotspots form 3D clusters over ∼200 kb chromosomal regions. Here, we investigated LinE configurations and distributions in live cells using super-resolution fluorescence microscopy. We found LinEs form two chromosomal structures, dot-like and linear structures, in both zygotic and azygotic meiosis. Dot-like LinE structures appeared around the time of meiotic DNA replication, underwent dotty-to-linear-to-dotty configurational transitions, and disassembled before the first meiotic division. DSB formation and repair did not detectably influence LinE structure formation, but failure of DSB formation delayed disassembly. Recombination-deficient LinE missense mutants formed dot-like but not linear LinE structures. Our quantitative study reveals a transient form of LinE structures and suggests a novel role for LinE proteins in regulating meiotic events, such as DSB repair. We discuss the relation of LinEs and the synaptonemal complex in other species.

Study of the RBE depth dependence of protons using a damage diagnostic algorithm and its application in proton therapy based on Monte Carlo simulation

The present study aimed to calculate the yields of DNA breaks and the variation of relative biological effectiveness (RBE) at different depths for protons using Geant4-DNA. For this purpose, an atomic model of DNA and a DNA damage classification matrix were used to calculate different DNA break yields for 62-MeV protons. As the reference radiation, the secondary electron spectrum produced by 60Co was evaluated. This helped to calculate the SSB and DSB yields. Moreover, RBE was found to be between 1.1 at the first point and 1.51 in the Bragg peak region. In this region, it was 37% greater than the 5-mm depth in the plateau region. Considering different threshold energies, the energy deposition at 10.79 eV had the most contribution to the total damage. As the results suggested, the depth dependence of RBE should be taken into account for proton therapy. It was also found that DNA break yields significantly depend on the threshold energy value.

HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP-ribose modifications

PARP1 and PARP2 produce poly(ADP-ribose) in response to DNA breaks. HPF1 regulates PARP1/2 catalytic output, most notably permitting serine modification with ADP-ribose. However, PARP1 is substantially more abundant in cells than HPF1, challenging whether HPF1 can pervasively modulate PARP1. Here, we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates/dissociates from multiple PARP1 molecules, initiating serine modification before modification initiates on glutamate/aspartate, and accelerating initiation to be more comparable to elongation reactions forming poly(ADP-ribose). This “hit and run” mechanism ensures HPF1 contributions to PARP1/2 during initiation do not persist and interfere with PAR chain elongation. We provide structural insights into HPF1/PARP1 assembled on a DNA break, and assess HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of serine-ADP-ribose modification in cells and the efficiency of serine-ADP-ribose modification required for an acute DNA damage response.

Single-Strand Breaks at Genome Initiate FLT3-ITD Formation

The fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is the most common recurrent mutation in acute myeloid leukemia (AML). FLT3-ITD varies in size from 3 to over 200 bp, resulting in elongation of juxtamembrane domain coded by exon 14 and constitutive kinase activation. The FLT3-ITD is a poor prognostic marker found in 20-30% of AML. However, molecular mechanisms underlying ITD formation are remained to be elucidated. We have analyzed FLT3-ITD mutation-positive AML cases using next-generation sequencing (NGS) and speculated that DNA breakage initiates ITD formation. We developed artificial FLT3-ITD formation assay using CRISPR/Cas9 system. First, genomic DNA from 25 cases with FLT3-ITD mutation-positive AML was used to PCR-amplify the ITD cluster region (ICR; FLT3 exon 14-15) and sequenced by NGS. We extracted more than 3 bp of deletion and insertion. Total 139 independent ITD sequences were identified at varied variant allele frequency (VAF) (0.0005%-45.7%). Each case had 1 to 13 ITDs (median 4 clones). The length of ITDs was 6 to 201 bp (median 48 bp) and 135 (97.1%) unique ITDs showed length with a multiple of 3 bp. In addition, we found 32 clones with Complex ITD, which is considered to have multiple ITD events. Simple FLT3-ITD showed consecutive two repeated sequences with or without filler sequence between the repeated sequences (Fig. Ai). However, some clones showed three or four repetitive sequences (Fig. Aii, Aiii). Furthermore, some clones had sequential second ITD including parts of the first ITD (Fig. Aiv), and some clones had two ITDs at adjacent or distant locations (Fig. Av). These "Complex ITD" was seen in 18 (72%) cases and always accompanying originated "Simple ITD" clones with higher VAF. Total 59 independent deletion sequences were identified in 24 out of 25 (96%) cases. Length of deletion of ICR is 3 to 204 bp (median 4.5 bp). Deletion clone is always rare clone which had a few reads. Non-ITD insertions were found only in 5 clones. The presence of multiple ITD clones in a single case, Complex ITD clones, and deletion clones in the ICR suggest that the ICR is prone to genomic damage, and the mutation process is ongoing in each AML case creating various ITD/deletions. Based on the observation of clinical samples, we investigated whether artificially induced DNA break at ICR repaired as ITDs in the human cell line. The FLT3 ICR was TA-cloned into the pGEM-T easy vector. FLT3 exon 14-targeted guide RNA and Cas9 protein were incubated with the vector in vitro and transfected to HEK293T cells. We compared conventional Cas9 inducing double-strand break (DSB) and Cas9-nickase inducing single-strand break (SSB) to determine the efficacy of ITD formation depending on the different DNA break modes. Genomic DNA was extracted from transfected HEK293T and successfully repaired ICR was amplified with primers annealing to pGEM-T easy vector flanking the cloning site. The amplified PCR product was analyzed by NGS with a 250 bp pair-end read. We extracted 545 and 353 miss repair events from DSB and SSB experiments respectively. The DSB of ICR was repaired as ITDs 1.1%, non-ITD insertions 7.5%, and deletions 91.4% (Fig. B). On the other hand, SSB of ICR was repaired as ITDs 7.3%, non-ITD insertions 1.4%, and deletions 91.2% (Fig. B). Within insertion event, ITD frequency was significantly higher in SSB compared to DSB. (p<0.001; chi-square test). Length of ITDs were 3 to 13 bp (median 3.5 bp) in DSB and 3 to 75 bp (median 28 bp) in SSB experiment (Fig. C). The ITD formed by the SSB was significantly longer than that formed by the DSB (p=0.0013; Mann-Whitney U test) and similar to observation in clinical simple ITD (Fig. C, D). Furthermore, we induced in-vivo SSB at endogenous FLT3 exon14 in HEK293T cells and successfully detected in situ ITDs. Using CRISPR induced SSB, we might develop a cell line with artificial FLT3-ITD which would contribute to deepen understanding of FLT3 biology. SSBs at FLT3 ICR could be a key initiator of FLT3-ITD formation. Progress in understanding the molecular mechanism of FLT3-ITD formation may lead to the development of therapeutic agents in the future. Figure 1 Figure 1. Disclosures Nakagawa: AbbVie GK: Research Funding; Takeda Pharmaceutical Company: Research Funding. Kondo: Astellas Pharma Inc.: Consultancy, Honoraria; Sanwa Kagaku Kenkyusho CO.,LTD: Consultancy; Sumitomo Dainippon Pharma: Honoraria; Bristol-Myers Squibb Company: Honoraria; Novartis Pharma KK: Honoraria; Otsuka Pharmaceutical: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Pfizer: Honoraria. Teshima: Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Company: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co.,Ltd.: Honoraria, Research Funding; Merck Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Fuji pharma CO.,Ltd: Research Funding; Pfizer Inc.: Honoraria; TEIJIN PHARMA Limited: Research Funding; Gentium/Jazz Pharmaceuticals: Consultancy; Novartis International AG: Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Janssen Pharmaceutical K.K.: Other; Bristol Myers Squibb: Honoraria; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Sanofi S.A.: Research Funding.

Genome-wide mapping implicates R-loops in lineage-specific processes and formation of DNA break hotspots in neural stem/progenitor cells

ABSTRACTRecent work has revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes. However, the mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of DNA:RNA hybrid structures called “R-loops” in primary neural stem/progenitor cells in order to assess their contribution to the different classes of DNA break “hotspots”. We report that R-loops in neural stem/progenitor cells are associated primarily with transcribed regions that replicate early and genes that show GC skew in their promoter region. Surprisingly, the majority of genes with recurrent DSB clusters in long, neural genes does not show substantial R-loop accumulation. We implicate R-loops in promoter-proximal DNA break formation in highly transcribed, early replicating regions and conclude that R-loops are not a driver of recurrent double-strand break cluster formation in most long, neural genes. Together, our study provides an understanding of how R-loops may contribute to DNA break hotspots and affect lineage-specific processes in neural stem/progenitor cells.

The Intra- and Extra-Telomeric Role of TRF2 in the DNA Damage Response

Telomere repeat binding factor 2 (TRF2) has a well-known function at the telomeres, which acts to protect the telomere end from being recognized as a DNA break or from unwanted recombination. This protection mechanism prevents DNA instability from mutation and subsequent severe diseases caused by the changes in DNA, such as cancer. Since TRF2 actively inhibits the DNA damage response factors from recognizing the telomere end as a DNA break, many more studies have also shown its interactions outside of the telomeres. However, very little has been discovered on the mechanisms involved in these interactions. This review aims to discuss the known function of TRF2 and its interaction with the DNA damage response (DDR) factors at both telomeric and non-telomeric regions. In this review, we will summarize recent progress and findings on the interactions between TRF2 and DDR factors at telomeres and outside of telomeres.

Small tandem DNA duplications result from CST-guided Pol α-primase action at DNA break termini

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3′ ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3′ tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3′ overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.

Disruption of a ∼23-24 nucleotide small RNA pathway elevates DNA damage responses in Tetrahymena thermophila

Endogenous RNA interference (RNAi) pathways regulate a wide range of cellular processes in diverse eukaryotes, yet in the ciliated eukaryote, Tetrahymena thermophila, the cellular purpose of RNAi pathways that generate ∼23-24 nucleotide (nt) small (s)RNAs has remained unknown. Here, we investigated the phenotypic and gene expression impacts on vegetatively growing cells when genes involved in ∼23-24 nt sRNA biogenesis are disrupted. We observed slower proliferation and increased expression of genes involved in DNA metabolism and chromosome organization and maintenance in sRNA biogenesis mutants RSP1Δ, RDN2Δ, and RDF2Δ. In addition, RSP1Δ and RDN2Δ cells frequently exhibited enlarged chromatin extrusion bodies, which are non-nuclear, DNA-containing structures that may be akin to mammalian micronuclei. Expression of homologous recombination factor Rad51 was specifically elevated in RSP1Δ and RDN2Δ strains, with Rad51 and double-stranded DNA break (DSB) marker γ-H2A.X localized to discrete macronuclear foci. In addition, an increase in Rad51 and γ-H2A.X foci were also found in knockouts of TWI8, a macronucleus-localized PIWI protein. Together, our findings suggest that an evolutionarily conserved role for RNAi pathways in maintaining genome integrity may be extended even to the early branching eukaryotic lineage that gave rise to Tetrahymena thermophila.