Subsequent Nonhomologous End-joining Research Articles

CRISPR Nickase-Mediated Base Editing in Yeast.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has enabled efficient, markerless genome editing in a wide range of organisms. However, there is an off-target effect and a limit to the area of precise editing. Bases that can be precisely edited are limited to within the 20-base pair gRNA-targeting site and protospacer adjacent motif (PAM) sequence. We have developed a CRISPR nickase system that can perform a precise genome-wide base editing in Saccharomyces cerevisiae using a single Cas9 nickase. This system can precisely edit a broader genomic region by the avoidance of double-strand break (DSB) and subsequent non-homologous end joining (NHEJ). Furthermore, unintended mutations were not found at off-target sites in this system. In combination with yeast gap repair cloning, precise genome editing of yeast cells can be performed in 5days. Here, we describe the methods for precise and convenient genome editing using this novel CRISPR nickase system.

The WD40 domain of FBXW7 is a poly(ADP-ribose)-binding domain that mediates the early DNA damage response.

FBXW7, a classic tumor suppressor, is a substrate recognition subunit of the Skp1-cullin-F-box (SCF) ubiquitin ligase that targets oncoproteins for ubiquitination and degradation. We recently found that FBXW7 is recruited to DNA damage sites to facilitate nonhomologous end-joining (NHEJ). The detailed underlying molecular mechanism, however, remains elusive. Here we report that the WD40 domain of FBXW7, which is responsible for substrate binding and frequently mutated in human cancers, binds to poly(ADP-ribose) (PAR) immediately following DNA damage and mediates rapid recruitment of FBXW7 to DNA damage sites, whereas ATM-mediated FBXW7 phosphorylation promotes its retention at DNA damage sites. Cancer-associated arginine mutations in the WD40 domain (R465H, R479Q and R505C) abolish both FBXW7 interaction with PAR and recruitment to DNA damage sites, causing inhibition of XRCC4 polyubiquitination and NHEJ. Furthermore, inhibition or silencing of poly(ADP-ribose) polymerase 1 (PARP1) inhibits PAR-mediated recruitment of FBXW7 to the DNA damage sites. Taken together, our study demonstrates that the WD40 domain of FBXW7 is a novel PAR-binding motif that facilitates early recruitment of FBXW7 to DNA damage sites for subsequent NHEJ repair. Abrogation of this ability seen in cancer-derived FBXW7 mutations provides a molecular mechanism for defective DNA repair, eventually leading to genome instability.

CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles

Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' overhang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9:gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.

TDP1 is required for efficient non-homologous end joining in human cells

Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3′ and 5′ terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation. Using a chromosomally-integrated NHEJ reporter substrate to compare end joining between wild type and TDP1-KO cells, we found that TDP1-KO cells have a 5-fold reduced ability to repair I-SceI-generated DSBs. Extracts prepared from TDP1-KO cells had reduced NHEJ activity in vitro, as compared to extracts from wild type cells. Analysis of end-joining junctions showed that TDP1 deficiency reduced end-joining fidelity, with a significant increase in insertion events, similar to previous observations in yeast. It has been reported that phosphorylation of TDP1 serine 81 (TDP1-S81) by ATM and DNA-PK stabilizes TDP1 and recruits TDP1 to sites of DNA damage. We found that end joining in TDP1-KO cells was partially restored by the non-phosphorylatable mutant TDP1-S81A, but not by the phosphomimetic TDP1-S81E. We previously reported that TDP1 physically interacted with XLF. In this study, we found that XLF binding by TDP1 was reduced 2-fold by the S81A mutation, and 10-fold by the S81E phosphomimetic mutation. Our results demonstrate a novel role for TDP1 in NHEJ in human cells. We hypothesize that TDP1 participation in human NHEJ is mediated by interaction with XLF, and that TDP1-XLF interactions and subsequent NHEJ events are regulated by phosphorylation of TDP1-S81.

Transposable elements and genomics

Once described as selfish DNA, transposable elements (TEs) have been revisited due to a plethora of new genomic information. TEs are considered indispensable genomic treasures during eukaryotic species evolution and supply raw materials for evolution by creating novel variation and allelic diversity (Volff 2006; Oliver et al. 2013). TEs are found ubiquitously with high copies in nearly all eukaryotic species. TEs are often present in major genomic denizens in some species by occupying more than half of the genome, contributing to the C-value paradox in eukaryotes. In this issue, six articles are published describing several aspects of transposable elements. Four review articles and two research articles are presented in this special issue. TEs are classified into class I TEs and class II TEs based on transposition mechanisms. Class I TEs are retrotransposons that retrotranspose via RNA intermediate semi-conservative manner. Class II TEs transpose via DNA intermediate conservative manner. Rajput (2015) described class I retrotransposons as the intrinsic genomic evolutionist as ‘‘Retrotransposons: the intrinsic genomic evolutionist’’. He reviewed essential information on retrotransposons. Retrotransposons are major agents in genome evolution and diversity by inducing rearrangements and restructuring genomes. Retrotransposons affect many aspects of biological organization, from gene regulation to genome obesity, which are critical in shaping current genomes. He also discusses the similarity between retrotransposons and endo-retroviruses, bringing the retrotransposons into a closer relationship to understand phylogenetics. Bae et al. contributed ‘‘Biological changes of transposable elements by radiation’’ (Bae et al. 2015). Human endogenous retroviruses (HERV) are a group of LTR (long terminal repeat) retrotransposons in the human genome. Transcriptionally-active TEs can move around in the genome and cause genome instability. HERV insertion into critical genes may lead to the progression of some diseases, including cancer. These authors reviewed cases of human disease due to HERV insertion. Radiation therapy manifests diverse, complex, and multicellular responses, including unmasking epigenetic suppression that switches silenced TEs into active ones. In the review, the authors summarized the recent understanding on radiationinduced DNA double-strand breaks (DSBs) and subsequent non-homologous end joining (NHEJ) repair. They also presented their recent results on activation of L1 elements by radiation in cancer cells, in which the methylation level of L1 was increased by relatively low radiation doses. Based on these results, the authors proposed that radiation exposure may lead directly to mutation as well as to methylation changes in TEs, which are considered important factors in cancer radiation therapy. Lee et al. contributed ‘‘Composition and evolutionary importance of transposable elements in humans and primates’’ (Lee et al. 2015). Primates, including humans, are not immune to the evolutionary impact of TEs on their genomes. Approximately half of the sequences of primate genomes are TErelated sequences. In their review, comparisons of TE constitution among primate species are extensively explained, and TE dispersions in the genome were analyzed between human and primates. These authors proposed that TE-driven structural variations are a major force in primate evolution. Roy et al. examined TEs in different aspects of molecular markers in ‘‘Marker utility of transposable elements for plant genetics, breeding, and N.-S. Kim (&) Department of Molecular Bioscience, Institute of Bioscience and Biotechnology, Kangwon National University, Chuncheon 200-701, Korea e-mail: kimnamsu@kangwon.ac.kr; nsk6472@yahoo.ca