OBJECTIVETo construct DNA double-strand break (DSB) repair protein hKu70 deficient cell strain and to observe its biological characters for studying the functions of hKu70 gene and the effects of occupational harmfulness factors on DSB repair.

METHODSHuman lung fibroblasts (HLF) were transfected with the eukaryotic expression plasmids of hKu70 gene antisense RNA (pEGFP-C1-K) to construct hKu70 protein deficient cells (named as "HLFK"). The protein expression levels of hKu70 gene in HLFC and HLFK were detected by the Western blotting to estimate the effects of antisense inhibition. Morphology, growth character and growth status in soft agar of transfected HLFK were observed.

RESULTSpEGFP-C1-K vector was successfully expressed in HLF. The protein expression level of hKu70 gene in HLFK was decreased by 42% as compared with that in HLFC. No obvious changes of the biologic characters were observed in HLFK.

CONCLUSIONThe hKu70 protein deficient cell strain was successfully constructed. The hKu70 protein deficiency alone didn't induce obvious changes of the biological characters in HLFK.

Antigens, Nuclear ; analysis ; Cell Division ; DNA Damage ; DNA Helicases ; DNA Repair ; DNA-Binding Proteins ; analysis ; deficiency ; Humans ; Ku Autoantigen ; RNA, Antisense ; Transfection

A common DNA polymerase can replicate DNA which functions normally. However, if DNA suffers damage, the genome can not be replicated by a common DNA polymerase because DNA lesions will block the replication apparatus. Another kind of DNA polymerases in organism, Y-family DNA polymerases which is also called translesion synthesis (TLS) polymerases, can deal with this problem. Their main functions are bypassing the lesions in DNA, replicating the genome and saving the dying cells. This thesis presents a historical review of the literature pertinent to the structure, functions and roles of Y-family DNA polymerases.
DNA Damage ; DNA Repair ; DNA Replication ; DNA-Directed DNA Polymerase ; classification ; metabolism ; physiology ; Humans ; Mutagenesis ; Mutagens ; Proliferating Cell Nuclear Antigen ; genetics

The Rad1 gene is evolutionarily conserved from yeast to human. The fission yeast Schizosaccharomyces pombe Rad1 ortholog promotes cell survival against DNA damage and is required for G(2)/M checkpoint activation. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad1, the mouse ortholog of this gene, were created to evaluate its function in mammalian cells. Mrad1 (-/-) ES cells were highly sensitive to ultraviolet-light (UV light), hydroxyurea (HU) and gamma rays, and were defective in G(2)/M as well as S/M checkpoints. These data indicate that Mrad1 is required for repairing DNA lesions induced by UV-light, HU and gamma rays, and for mediating G(2)/M and S/M checkpoint controls. We further demonstrated that Mrad1 plays an important role in homologous recombination repair (HRR) in ES cells, but a minor HRR role in differentiated mouse cells.
Animals ; Cell Division ; Cell Proliferation ; DNA Damage ; DNA Repair ; Embryonic Stem Cells ; metabolism ; Exonucleases ; genetics ; metabolism ; physiology ; G2 Phase ; Gamma Rays ; Gene Deletion ; Hydroxyurea ; pharmacology ; Mice ; Ultraviolet Rays

DNA damage accumulates in cells as a result of exposure to exogenous agents such as benzopyrene, cigarette smoke, ultraviolet light, X-ray, and endogenous chemicals including reactive oxygen species produced from normal metabolic byproducts. DNA damage can also occur during aberrant DNA processing reactions such as DNA replication, recombination, and repair. The major of DNA damage affects the primary structure of the double helix; that is, the bases are chemically modified. These modification can disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. DNA repair genes and proteins scan the global genome to detect and remove DNA damage and damage to single nucleotides. Direct reversal of DNA damage, base excision repair, double strand break. DNA repair are known relevant DNA repair mechanisms. Four different mechanisms are distinguished within excision repair: direct reversal, base excision repair, nucleotide excision repair, and mismatch repair. Genetic variation in DNA repair genes can modulate DNA repair capacity and alter cancer risk. The instability of a cell to properly regulate its proliferation in the presence of DNA damage increase risk of gene mutation and carcinogenesis. This article aimed to review mechanism of excision repair and to understand the relationship between genetic variation of excision repair genes and head and neck cancer.
DNA ; DNA Damage ; DNA Mismatch Repair ; DNA Repair ; DNA Replication ; Genetic Variation ; Genome ; Head ; Head and Neck Neoplasms ; Nucleotides ; Proteins ; Reactive Oxygen Species ; Recombination, Genetic ; Smoke ; Tobacco Products ; Ultraviolet Rays

PURPOSE: Heterochromatin protein 1gamma (HP1gamma) interacts with chromosomes by binding to lysine 9-methylated histone H3 or DNA/RNA. HP1gamma is involved in various biological processes. The purpose of this study is to gain an understanding of how HP1gamma functions in these processes by identifying HP1gamma-binding proteins using mass spectrometry. MATERIALS AND METHODS: We performed affinity purification of HP1gamma-binding proteins using G1/S phase or prometaphase HEK293T cell lysates that transiently express mock or FLAG-HP1gamma. Coomassie staining was performed for HP1gamma-binding complexes, using cell lysates prepared by affinity chromatography FLAG-agarose beads, and the bands were digested and then analyzed using a mass spectrometry. RESULTS: We identified 99 HP1gamma-binding proteins with diverse cellular functions, including spliceosome, regulation of the actin cytoskeleton, tight junction, pathogenic Escherichia coli infection, mammalian target of rapamycin signaling pathway, nucleotide excision repair, DNA replication, homologous recombination, and mismatch repair. CONCLUSION: Our results suggested that HP1gamma is functionally active in DNA damage response via protein-protein interaction.
Actin Cytoskeleton ; Biological Processes ; Chromatography, Affinity ; DNA Damage* ; DNA Mismatch Repair ; DNA Repair ; DNA Replication ; DNA* ; Escherichia coli Infections ; Heterochromatin* ; Histones ; Homologous Recombination ; Lysine ; Mass Spectrometry ; Prometaphase ; Sirolimus ; Spliceosomes ; Tight Junctions

Animals ; DNA Damage ; DNA Repair ; DNA-Activated Protein Kinase ; Humans

Animals ; DNA Damage ; DNA Repair ; DNA, Neoplasm ; Humans

53BP1 is an important genome stability regulator, which protects cells against double-strand breaks. Following DNA damage, 53BP1 is rapidly recruited to sites of DNA breakage, along with other DNA damage response proteins, including gamma-H2AX, MDC1, and BRCA1. The recruitment of 53BP1 requires a tandem Tudor fold which associates with methylated histones H3 and H4. It has already been determined that the majority of DNA damage response proteins are phosphorylated by ATM and/or ATR after DNA damage, and then recruited to the break sites. 53BP1 is also phosphorylated at several sites, like other proteins after DNA damage, but this phosphorylation is not critically relevant to recruitment or repair processes. In this study, we evaluated the functions of phosphor-53BP1 and the role of the BRCT domain of 53BP1 in DNA repair. From our data, we were able to detect differences in the phosphorylation patterns in Ser25 and Ser1778 of 53BP1 after neocarzinostatin-induced DNA damage. Furthermore, the foci formation patterns in both phosphorylation sites of 53BP1 also evidenced sizeable differences following DNA damage. From our results, we concluded that each phosphoryaltion site of 53BP1 performs different roles, and Ser1778 is more important than Ser25 in the process of DNA repair.
DNA ; DNA Damage ; DNA Repair ; Genomic Instability ; Histones ; Phosphorylation ; Proteins

DNA ; DNA Damage ; DNA Repair ; Fanconi Anemia/genetics* ; Humans

Eukaryotic organisms constantly face a wide range of internal and external factors that cause damage to their DNA. Failure to accurately and efficiently repair these DNA lesions can result in genomic instability and the development of tumors (Canela et al., 2017). Among the various forms of DNA damage, DNA double-strand breaks (DSBs) are particularly harmful. Two major pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), are primarily responsible for repairing DSBs (Katsuki et al., 2020; Li and Yuan, 2021; Zhang and Gong, 2021; Xiang et al., 2023). NHEJ is an error-prone repair mechanism that simply joins the broken ends together (Blunt et al., 1995; Hartley et al., 1995). In contrast, HR is a precise repair process. It involves multiple proteins in eukaryotic cells, with the RAD51 recombinase being the key player, which is analogous to bacterial recombinase A (RecA) (Shinohara et al., 1992). The central event in HR is the formation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments that facilitate homology search and DNA strand invasion, ultimately leading to the initiation of repair synthesis (Miné et al., 2007; Hilario et al., 2009; Ma et al., 2017).
Recombinational DNA Repair ; DNA-Binding Proteins/metabolism* ; DNA Repair ; DNA Damage ; DNA