Enzymatic repair of UV-damaged DNA in mammalian cells: perspectives

1977 ◽  
Vol 46 (2) ◽  
pp. 147-148
Keyword(s):  
Mammalian Cells ◽  
Damaged Dna

scholarly journals Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells

2020 ◽  
Vol 21 (20) ◽  
pp. 7564
Author(s):  
Dipon Das ◽  
Molly L. Bristol ◽  
Pietro Pichierri ◽  
Iain M. Morgan
Keyword(s):  
Dna Replication ◽  
Viral Genome ◽  
Mammalian Cells ◽  
Human Papillomaviruses ◽  
Lacz Gene ◽  
Dna Templates ◽  
Protein Protein Interaction ◽  
Dna Replication And Repair ◽  
Cellular Components ◽  
Damaged Dna

Human papillomaviruses have 8kbp DNA episomal genomes that replicate autonomously from host DNA. During initial infection, the virus increases its copy number to 20–50 copies per cell, causing torsional stress on the replicating DNA. This activates the DNA damage response (DDR) and HPV replicates its genome, at least in part, using homologous recombination. An active DDR is on throughout the HPV life cycle. Two viral proteins are required for replication of the viral genome; E2 binds to 12bp palindromic sequences around the A/T rich origin of replication and recruits the viral helicase E1 via a protein–protein interaction. E1 forms a di-hexameric complex that replicates the viral genome in association with host factors. Transient replication assays following transfection with E1–E2 expression plasmids, along with an origin containing plasmid, allow monitoring of E1-E2 replication activity. Incorporating a bacterial lacZ gene into the origin plasmid allows for the determination of replication fidelity. Here we describe how we exploited this system to investigate replication and repair in mammalian cells, including using damaged DNA templates. We propose that this system has the potential to enhance the understanding of cellular components involved in DNA replication and repair.

scholarly journals Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

2021 ◽  
Author(s):  
Yasunori Horikoshi ◽  
Hiroki Shima ◽  
Wataru Kobayashi ◽  
Jiying Sun ◽  
Volker J Schmid ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Super Resolution ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Sister Chromatids ◽  
Super Resolution Microscopy ◽  
Damaged Dna

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1

1984 ◽  
Vol 4 (2) ◽  
pp. 324-328
Author(s):  
C Dinsart ◽  
J J Cornelis ◽  
B Klein ◽  
A J van der Eb ◽  
J Rommelaere
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Virus ◽  
Double Stranded Dna ◽  
Mutator Activity ◽  
Calf Thymus ◽  
Circular Dnas ◽  
Uv Lesions ◽  
Damaged Dna

Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

scholarly journals Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1.

1984 ◽  
Vol 4 (2) ◽  
pp. 324-328 ◽  
Author(s):  
C Dinsart ◽  
J J Cornelis ◽  
B Klein ◽  
A J van der Eb ◽  
J Rommelaere
Keyword(s):  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Virus ◽  
Double Stranded Dna ◽  
Mutator Activity ◽  
Calf Thymus ◽  
Circular Dnas ◽  
Uv Lesions ◽  
Damaged Dna

Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

scholarly journals PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

2008 ◽  
Vol 183 (7) ◽  
pp. 1203-1212 ◽  
Author(s):  
Kazuto Sugimura ◽  
Shin-ichiro Takebayashi ◽  
Hiroshi Taguchi ◽  
Shunichi Takeda ◽  
Katsuzumi Okumura
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Parp Inhibitor ◽  
Small Interfering Rnas ◽  
Parp 1 ◽  
Dt40 Cells ◽  
Damaged Dna

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.

scholarly journals Characterization of a DNA-damage-recognition protein from F9 teratocarcinoma cells, which is inducible by retinoic acid and cyclic AMP

1993 ◽  
Vol 290 (1) ◽  
pp. 129-134 ◽  
Author(s):  
C C K Chao ◽  
N K Sun ◽  
S Lin-Chao
Keyword(s):  
Dna Damage ◽  
Retinoic Acid ◽  
Cyclic Amp ◽  
Mammalian Cells ◽  
Nuclear Protein ◽  
Binding Activity ◽  
Damage Recognition ◽  
Nuclear Extracts ◽  
Recognition Protein ◽  
Damaged Dna

A nuclear protein that recognizes u.v.-damaged DNA was detected in extracts from murine F9 embryonic stem cells using a DNA-binding assay. The nuclear-protein-binding activity was increased in cells after treatment with retinoic acid/dibutyryl cyclic AMP (dbcAMP), with optimum induction at 6 days. In vitro treatment of nuclear extracts with agents that affect protein conformation (such as urea, Nonidet P40 and Ca2+) slightly modulated the damage-recognition activity. Furthermore, treatment of nuclear extracts with phosphatase dramatically inhibited the binding activity. In addition, damaged-DNA recognition of the nuclear extracts was effectively inhibited by damaged double- and single-stranded DNA. The expression of the nuclear protein with similar characteristics was abundant in HeLa cells and was increased in drug- or u.v.-resistant cells. The findings suggest that the recognition of a u.v.-DNA adduct is modulated, at least in part, by an activity that is induced during retinoic acid/dbcAMP-induced differentiation. These results also imply that the identified damage-recognition protein may be important for the sensitivity or resistance of mammalian cells to DNA damage.

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