Search

Extrachromosomal circular DNA (eccDNA) are present within all eukaryotic organisms and actively contribute to gene expression changes. MicroDNA (200-1000bp) are the most abundant type of eccDNA and can amplify tRNA, microRNA, and novel si-like RNA sequences. Due to the heterogeneity of microDNA and the limited technology to directly quantify circular DNA molecules, the specific DNA repair pathways that contribute to microDNA formation have not been fully elucidated. Using a sensitive and quantitative assay that quantifies eight known abundant microDNA, we report that microDNA levels are dependent on resection after double-strand DNA break (DSB) and repair by Microhomology Mediated End Joining (MMEJ). Further, repair of DSB without resection by canonical Non-Homologous End Joining (c-NHEJ) diminishes microDNA formation. MicroDNA levels are induced locally even by a single site-directed DSB, suggesting that excision of genomic DNA by two closely spaced DSB is not necessary for microDNA formation. Consistent with all this, microDNA levels accumulate as cells undergo replication in S-phase, when DNA breaks and repair are elevated, and microDNA levels are decreased if DNA synthesis is prevented. Thus, formation of microDNA occurs during the repair of endogenous or induced DNA breaks by resection-based DNA repair pathways.

Accumulating evidence suggests participation of RNA-binding proteins with intrinsically disordered domains (IDPs) in the DNA damage response (DDR). These IDPs form liquid compartments at DNA damage sites in a poly(ADP ribose) (PAR)-dependent manner. However, it is greatly unknown how the IDPs are involved in DDR. We have shown previously that one of the IDPs RBM14 is required for the canonical nonhomologous end joining (cNHEJ). Here we show that RBM14 is recruited to DNA damage sites in a PARP- and RNA polymerase II (RNAPII)-dependent manner. Both KU and RBM14 are required for RNAPII-dependent generation of RNA:DNA hybrids at DNA damage sites. In fact, RBM14 binds to RNA:DNA hybrids. Furthermore, RNA:DNA hybrids and RNAPII are detected at gene-coding as well as at intergenic areas when double-strand breaks (DSBs) are induced. We propose that the cNHEJ pathway utilizes damage-induced transcription and intrinsically disordered protein RBM14 for efficient repair of DSBs.

Objective To understand role of barrier molecules in melanomas. Background We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. Methods FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. Results Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. Conclusions FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.

Extrachromosomal circular DNAs (eccDNA) are widespread in normal and cancer cells and are known to amplify oncogenic genes. However, the mechanisms that form eccDNA have never been fully elucidated due to the complex interactions of DNA repair pathways and lack of a method to quantify eccDNA abundance. Through the development of a sensitive and quantitative assay for eccDNA we show that the formation of eccDNA is through resection dependent repair of double-strand DNA breaks, especially micro-homology mediated end joining, and through mismatch repair. The most significant decreases in eccDNA levels occurred in cells lacking PARP1, POLQ, NBS1, RAD54, and FAN1. Further, a significant increase in eccDNA occurred in cells lacking c-NHEJ proteins DNA-PKcs, XRCC4, XLF, LIG4 and 53BP1. This suggests that when alt-NHEJ pathways are utilized to repair DNA breaks by necessity, the formation of eccDNA is increased. Induced and site-directed double-strand DNA breaks increase eccDNA formation, even from a single break. Additionally, we find that eccDNA levels accumulate as cells undergo replication in S-phase and that levels of eccDNA are decreased if DNA synthesis is prevented. Together, these results show that the bulk of eccDNA form by resection based alt-NHEJ pathways, especially during DNA replication and the repair of double-strand breaks.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.

F-box proteins are substrate receptors of the SCF (SKP1-Cullin 1-F-box protein) E3 ubiquitin ligase that play important roles in a number of physiological processes and activities. Through their ability to assemble distinct E3 ubiquitin ligases and target key regulators of cellular activities for ubiquitylation and degradation, this versatile group of proteins is able to regulate the abundance of cellular proteins whose deregulated expression or activity contributes to disease. In this review, we describe the important roles of select F-box proteins in regulating cellular activities, the perturbation of which contributes to the initiation and progression of a number of human malignancies.