Search Results (6)

Various DNA damage checkpoint control mechanisms in eukaryotic cells help maintain genomic integrity. Among these, NBS1, a key component of the MRE11-RAD50-NBS1 (MRN) complex, is an essential protein involved in the DNA damage response (DDR). In this study, we discovered that DNA damage-binding protein 1 (DDB1) interacts with NBS1. DDB1 is a DDR sensor protein found in UV-induced DNA replication blocks. Through pull-down and immunoprecipitation assays conducted in Xenopus egg extracts and human cell lines, we demonstrated a specific interaction between NBS1 and DDB1. DDB1 was also found to associate with several proteins that interact with NBS1, including DNA topoisomerase 2-binding protein 1 (TopBP1) and Mediator of DNA damage checkpoint protein 1 (MDC1). Notably, the interaction between DDB1 and NBS1 is disrupted in MDC1-depleted egg extracts, indicating that MDC1 is necessary for this interaction. Furthermore, the depletion of DDB1 leads to increased Chk1 activation upon DNA damage. These novel findings regarding the interaction between NBS1 and DDB1 provide new insights into how DDB1 regulates DNA damage pathways. Full article

In plants, prolonged exposure to ultraviolet (UV) radiation causes harmful DNA lesions. Nucleotide excision repair (NER) is an important DNA repair mechanism that operates via two pathways: transcription coupled repair (TC-NER) and global genomic repair (GG-NER). In plants and mammals, TC-NER is initiated by the Cockayne Syndrome A and B (CSA/CSB) complex, whereas GG-NER is initiated by the Damaged DNA Binding protein 1/2 (DDB1/2) complex. In the yeast Saccharomyces cerevisiae (S. cerevisiae), GG-NER is initiated by the Radiation Sensitive 7 and 16, (RAD7/16) complex. Arabidopsis thaliana has two homologues of yeast RAD16, At1g05120 and At1g02670, which we named AtRAD16 and AtRAD16b, respectively. In this study, we characterized the roles of AtRAD16 and AtRAD16b. Arabidopsis rad16 and rad16b null mutants exhibited increased UV sensitivity. Moreover, AtRAD16 overexpression increased plant UV tolerance. Thus, AtRAD16 and AtRAD16b contribute to plant UV tolerance and growth. Additionally, we found physical interaction between AtRAD16 and AtRAD7. Thus, the Arabidopsis RAD7/16 complex is functional in plant NER. Furthermore, AtRAD16 makes a significant contribution to Arabidopsis UV tolerance compared to the DDB1/2 and the CSB pathways. This is the first time the role and interaction of DDB1/2, RAD7/16, and CSA/CSB components in a single system have been studied. Full article

Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins which work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases, resulting in abasic sites. Many DNA glycosylases are product-inhibited by binding to the abasic site more avidly than the damaged base. Traditionally, apurinic/apyrimidinic endonuclease 1, APE1, was believed to help turn over the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory, we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three- and five-fold. Moreover, we have shown that UV-DDB can assist chromatin decompaction, facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER. Full article

DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo. Full article

LAP2-Emerin-MAN1 (LEM) domain-containing proteins represent an abundant group of inner nuclear membrane proteins involved in diverse nuclear functions, but their functional redundancies remain unclear. Here, using the biotinylation-dependent proximity approach, we report proteome-wide comparative interactome analysis of the two structurally related LEM proteins MAN1 (LEMD3) and LEM2 (LEMD2), and the more distantly related emerin (EMD). While over 60% of the relatively small group of MAN1 and emerin interactors were also found in the LEM2 interactome, the latter included a large number of candidates (>85%) unique for LEM2. The interacting partners unique for emerin support and provide further insight into the previously reported role of emerin in centrosome positioning, and the MAN1-specific interactors suggest a role of MAN1 in ribonucleoprotein complex assembly. Interestingly, the LEM2-specific interactome contained several proteins of the nucleotide excision repair pathway. Accordingly, LEM2-depleted cells, but not MAN1- and emerin-depleted cells, showed impaired proliferation following ultraviolet-C (UV-C) irradiation and prolonged accumulation of γH2AX, similar to cells deficient in the nucleotide excision repair protein DNA damage-binding protein 1 (DDB1). These findings indicate impaired DNA damage repair in LEM2-depleted cells. Overall, this interactome study identifies new potential interaction partners of emerin, MAN1 and particularly LEM2, and describes a novel potential involvement of LEM2 in nucleotide excision repair at the nuclear periphery. Full article

Damaged DNA-binding protein 1 (DDB1) recruits nucleotide excision pathway proteins to form the UV-damaged DNA-binding protein complex and is required for DNA repair. DDB1 was reported to participate in apoptosis and chemoresistance regulation in several cancers. However, little is known about the function of DDB1 in pancreatic adenocarcinoma (PDAC). In this study, we reported that DDB1 functions as a tumor-promoting factor in PDAC by regulating cancer cell proliferation, epithelial-mesenchymal transition (EMT) and chemoresistance. Compared to normal pancreatic tissues, PDAC tissues had high expression levels of DDB1, and this high expression was positively correlated with poor prognosis. Furthermore, reductions in cell proliferation and EMT were observed in DDB1-deficient PDAC cell lines. Intriguingly, we also found that abrogation of DDB1 expression increased PDAC cell sensitivity to gemcitabine (GEM). Mechanistically, DDB1 knockdown was associated with an increase in deoxycytidine kinase expression in vivo and in vitro. In summary, our work demonstrated that DDB1 promotes PDAC progression and chemoresistance and may serve as a potential predictive marker and therapeutic target for PDAC treatment. Full article