Homologous Recombination-directed Repair Research Articles

Structure-specific nucleases in genome dynamics and strategies for targeting cancers.

Nucleases are a super family of enzymes that hydrolyze phosphodiester bonds present in genomes. They widely vary in substrates, causing differentiation in cleavage patterns and having a diversified role in maintaining genetic material. Through cellular evolution of prokaryotic to eukaryotic, nucleases become structure-specific in recognizing its own or foreign genomic DNA/RNA configurations as its substrates, including flaps, bubbles, and Holliday junctions. These special structural configurations are commonly found as intermediates in processes like DNA replication, repair, and recombination. The structure-specific nature and diversified functions make them essential to maintaining genome integrity and evolution in normal and cancer cells. In this article, we review their roles in various pathways, including Okazaki fragment maturation during DNA replication, end resection in homology-directed recombination repair of DNA double-strand breaks, DNA excision repair and apoptosis DNA fragmentation in response to exogeneous DNA damage, and HIV life cycle. As the nucleases serve as key points for the DNA dynamics, cellular apoptosis, and cancer cell survival pathways, we discuss the efforts in the field in developing the therapeutic regimens, taking advantage of recently available knowledge of their diversified structures and functions.

Recent advances and applications of base editing systems

The CRISPR system is able to accomplish precise base editing in genomic DNA, but relies on the cellular homology-directed recombination repair pathway and is therefore extremely inefficient. Base editing is a new genome editing technique developed based on the CRISPR/Cas9 system. Two base editors (cytosine base editor and adenine base editor) were developed by fusing catalytically disabled nucleases with different necleobase deaminases. These two base editors are able to perform C>T (G>A) or A>G (T>C) transition without generating DNA double-stranded breaks. The base editing technique has been widely used in gene therapy, animal models construction, precision animal breeding and gene function analysis, providing a powerful tool for basic and applied research. This review summarized the development process, technical advantages, current applications, challenges and perspectives for base editing technique, aiming to help the readers better understand and use the base editing technique.

Functional deficiency of DNA repair gene EXO5 results in androgen-induced genomic instability and prostate tumorigenesis.

Germline mutations of DNA double-strand break (DSB) response and repair genes that drive tumorigenesis could be a major cause of prostate cancer (PCa) heritability. In this study, we demonstrated the role of novel exonuclease 5 (EXO5) gene in androgen-induced double strand breaks repair via homology-directed repair pathway and prostate tumorigenesis. Using whole-exome sequencing of samples from 20 PCa families, with three or more siblings diagnosed with metastatic PCa, we identified mutations in 31 genes involved in DSB response and repair. Among them, the L151P mutation in the exonuclease 5 (EXO5) gene was present in all affected siblings in three PCa families. We found two other EXO5 SNPs significantly associated with risk of PCa in cases-controls study from databases of genotype and phenotype (dbGaP), which are in linkage disequilibrium (D' = 1) with Exo5 L151P found in PCa family. The L151 residue is conserved across diverse species and its mutation is deleterious for protein functions, as demonstrated by our bioinformatics analyses. The L151P mutation impairs the DNA repair function of EXO5 due to loss of nuclease activity, as well as failure of nuclear localization. CRISPR elimination of EXO5 in a PCa cell line impaired homology-directed recombination repair (HDR) and caused androgen-induced genomic instability, as indicated by frequent occurrence of the oncogenic fusion transcript TMPRSS2-ERG. Genetic and functional validation of the EXO5 mutations indicated that EXO5 is a risk gene for prostate tumorigenesis, likely due to its functions in HDR.

TRAF6 mediates human DNA2 polyubiquitination and nuclear localization to maintain nuclear genome integrity.

The multifunctional human DNA2 (hDNA2) nuclease/helicase is required to process DNA ends for homology-directed recombination repair (HDR) and to counteract replication stress. To participate in these processes, hDNA2 must localize to the nucleus and be recruited to the replication or repair sites. However, because hDNA2 lacks the nuclear localization signal that is found in its yeast homolog, it is unclear how its migration into the nucleus is regulated during replication or in response to DNA damage. Here, we report that the E3 ligase TRAF6 binds to and mediates the K63-linked polyubiquitination of hDNA2, increasing the stability of hDNA2 and promoting its nuclear localization. Inhibiting TRAF6-mediated polyubiquitination abolishes the nuclear localization of hDNA2, consequently impairing DNA end resection and HDR. Thus, the current study reveals a mechanism for the regulation of hDNA2 localization and establishes that TRAF6-mediated hDNA2 ubiquitination activates DNA repair pathways to maintain nuclear genome integrity.

HDAC Inhibition Induces microRNA-182 Which Targets Rad51 Protein and Impairs Homologous Recombination Repair to Sensitize Cells to the Double Strand Break Inducing Nucleoside Analog, Sapacitabine in AML

Acute myelogenous leukemia (AML) is characterized by multiple genetic and epigenetic abnormalities including a profound dysregulation of microRNA expression.Effective clinical treatment of AML has largely depended on a class of antimetabolites - the nucleoside analogs. Of these, sapacitabine is a nucleoside analog prodrug that is in development for the therapy of AML. It is converted to its active metabolite 2-C-cyano-2-deoxy-1-β(-D-arabino-pentafuranosyl) cytosine (CNDAC), which interferes with DNA synthesis by initially causing a single stranded DNA break that is converted into a double strand break in the subsequent replicative cycle. Such double strand breaks are primarily repaired by the homologous recombination repair (HR) pathway. Consequently, efficient HR may offer a potential resistance mechanism to therapy with sapacitabine. Rad51 is a protein plays a critical role in HR, and high levels of Rad51 are linked to resistance to DNA damaging therapies.Histone deacetylases (HDACs) are chromatin modulating agents that decrease levels of acetylation of histones, repress gene expression. HDAC inhibitors (HDACis) function by modifying chromatin to epigenetically reverse gene silencing of coding and non-coding genes such as the microRNAs (miRs). miRs are endogenous noncoding RNAs 19-25 nucleotides in length that bind to complimentary sequences in target RNA to either destabilize it or prevent its transcription.In this study, we determined that determined primary AML blasts and cell lines express low levels of microRNA-182. Recruitment of HDAC1 and its co-repressors were linked to the epigenetic silencing of miR-182 in AML. Conversely, HDAC inhibition led to accumulation of activating chromatin modifications followed by the upregulation of miR-182 in AML blasts and cell lines. The HDACi-induced increases in miR-182 were linked to decreases in the levels of Rad51, an inhibition in the ability of cells to conduct homologous recombination repair as measured by the Homologous recombination directed repair (HDR) assay, persistent levels of DNA damage as measured by the levels of Ɣ-H2AX and sensitization to sapacitabine. We then mechanistically defined the relation between miR-182 and Rad51. Ectopic expression of miR-182 in AML cell lines identified that Rad51 was a target of miR-182. An assay with luciferase constructs bearing full length or mutated Rad51 3'UTR indentified that Rad51 was a direct target of miR-182. We also determined that ectopic expression of miR-182 attenuated the ability of AML cells to conduct homologus repair as measured by the Homologous recombination directed repair (HDR) assay which resulted in sensitizing AML cells to the cytotoxic action of CNDAC as measured by colony forming assays.In conclusion, our data show that HDAC inhibitors target Rad51 via miR-182 to compromise HR repair to result in higher levels of residual DNA damage and sensitize AML cells to double strand damaging agents such as CNDAC. DisclosuresNo relevant conflicts of interest to declare.

Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing.

BackgroundThe ability to modify the genome of any cell at a precise location has drastically improved with the recent discovery and implementation of CRISPR/Cas9 editing technology. However, the capacity to introduce specific directed changes at given loci is hampered by the fact that the major cellular repair pathway that occurs following Cas9-mediated DNA cleavage is the erroneous non-homologous end joining (NHEJ) pathway. Homology-directed recombination (HDR) is far less efficient than NHEJ and makes screening of clones containing directed changes time-consuming and labor-intensive.MethodsWe investigated the possibility of pharmacologically inhibiting DNA-PKcs, a key player in NHEJ, using small molecule inhibitors (NU7441 and KU-0060648), to ameliorate the rates of HDR repair events. These compounds were tested in a sensitive reporter assay capable of simultaneously informing on NHEJ and HDR, as well as on an endogenous gene targeted by Cas9.ResultsWe find that NU7441 and KU-0060648 reduce the frequency of NHEJ while increasing the rate of HDR following Cas9-mediated DNA cleavage.ConclusionsOur results identify two small molecules compatible for use with Cas9-editing technology to improve the frequency of HDR.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0215-6) contains supplementary material, which is available to authorized users.

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0153-5) contains supplementary material, which is available to authorized users.

RPA Mediates Recombination Repair During Replication Stress and Is Displaced from DNA by Checkpoint Signalling in Human Cells

The replication protein A (RPA) is involved in most, if not all, nuclear metabolism involving single-stranded DNA. Here, we show that RPA is involved in genome maintenance at stalled replication forks by the homologous recombination repair system in humans. Depletion of the RPA protein inhibited the formation of RAD51 nuclear foci after hydroxyurea-induced replication stalling leading to persistent unrepaired DNA double-strand breaks (DSBs). We demonstrate a direct role of RPA in homology directed recombination repair. We find that RPA is dispensable for checkpoint kinase 1 (Chk1) activation and that RPA directly binds RAD52 upon replication stress, suggesting a direct role in recombination repair. In addition we show that inhibition of Chk1 with UCN-01 decreases dissociation of RPA from the chromatin and inhibits association of RAD51 and RAD52 with DNA. Altogether, our data suggest a direct role of RPA in homologous recombination in assembly of the RAD51 and RAD52 proteins. Furthermore, our data suggest that replacement of RPA with the RAD51 and RAD52 proteins is affected by checkpoint signalling.

Essential Role for Nuclear PTEN in Maintaining Chromosomal Integrity

A broad spectrum of mutations in PTEN, encoding a lipid phosphatase that inactivates the P13-K/AKT pathway, is found associated with primary tumors. Some of these mutations occur outside the phosphatase domain, suggesting that additional activities of PTEN function in tumor suppression. We report a nuclear function for PTEN in controlling chromosomal integrity. Disruption of Pten leads to extensive centromere breakage and chromosomal translocations. PTEN was found localized at centromeres and physically associated with CENP-C, an integral component of the kinetochore. C-terminal PTEN mutants disrupt the association of PTEN with centromeres and cause centromeric instability. Furthermore, Pten null cells exhibit spontaneous DNA double-strand breaks (DSBs). We show that PTEN acts on chromatin and regulates expression of Rad51, which reduces the incidence of spontaneous DSBs. Our results demonstrate that PTEN plays a fundamental role in the maintenance of chromosomal stability through the physical interaction with centromeres and control of DNA repair. We propose that PTEN acts as a guardian of genome integrity.

BRCA1 Facilitates Microhomology-mediated End Joining of DNA Double Strand Breaks

BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.