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1.
DNA Repair (Amst) ; 128: 103525, 2023 Aug.
Article in English | MEDLINE | ID: mdl-37320956

ABSTRACT

Immunofluorescence imaging is a standard experimental tool for monitoring the response of cellular factors to DNA damage. Visualizing the recruitment of DNA Damage Response (DDR) components requires high affinity antibodies, which are generally available. In contrast, reagents for the display of the lesions that induce the response are far more limited. Consequently, DDR factor accumulation often serves as a surrogate for damage, without reporting the actual inducing structure. This limitation has practical implications given the importance of the response to DNA reactive drugs such as those used in cancer therapy. These include interstrand crosslink (ICL) forming compounds which are frequently employed clinically. Among them are the psoralens, natural products that form ICLs upon photoactivation and applied therapeutically since antiquity. However, despite multiple attempts, antibodies against psoralen ICLs have not been developed. To overcome this limitation, we developed a psoralen tagged with an antigen for which there are commercial antibodies. In this report we describe our application of the tagged psoralen in imaging experiments, and the unexpected discoveries they revealed.


Subject(s)
DNA Repair , Ficusin , Ficusin/pharmacology , Cross-Linking Reagents/pharmacology , DNA Damage , DNA
2.
J Phys Chem A ; 118(45): 10359-63, 2014 Nov 13.
Article in English | MEDLINE | ID: mdl-24912084

ABSTRACT

The effect of Mg(2+) cations on the electronic spectra and dynamics and efficiency of hole transport has been determined by means of femtosecond time-resolved transient absorption spectroscopy for DNA hairpins possessing stilbene electron acceptor and donor chromophores. The results are compared with those obtained previously for the same hairpins in the presence of Na(+) cations and for one hairpin with no added salt. Quantum yields and rate constants for charge separation are smaller in the presence of Mg(2+) than Na(+), the largest differences being observed for the hairpins with the largest number of base pairs. Slower charge separation is attributed to minor groove binding by Mg(2+), which results in a stiffer duplex structure rather than a change in ground state geometry. Reduction in the Na(+) concentration has little effect on either the dynamics or efficiency of hole transport.


Subject(s)
Cations/chemistry , DNA/chemistry , Magnesium/chemistry , Circular Dichroism , Inverted Repeat Sequences , Magnesium Chloride/chemistry , Nucleic Acid Conformation , Photochemical Processes , Sodium/chemistry , Sodium Chloride/chemistry , Spectrum Analysis , Stilbenes/chemistry , Ultraviolet Rays
3.
Methods Mol Biol ; 1114: 103-13, 2014.
Article in English | MEDLINE | ID: mdl-24557899

ABSTRACT

Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site. Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity.


Subject(s)
Gene Expression , Gene Targeting/methods , Oligonucleotides/genetics , Animals , CHO Cells , Cell Culture Techniques , Cell Line , Cricetulus , Electroporation , Humans , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Oligonucleotides/isolation & purification
4.
Mol Cell ; 52(3): 434-46, 2013 Nov 07.
Article in English | MEDLINE | ID: mdl-24207054

ABSTRACT

The replicative machinery encounters many impediments, some of which can be overcome by lesion bypass or replication restart pathways, leaving repair for a later time. However, interstrand crosslinks (ICLs), which preclude DNA unwinding, are considered absolute blocks to replication. Current models suggest that fork collisions, either from one or both sides of an ICL, initiate repair processes required for resumption of replication. To test these proposals, we developed a single-molecule technique for visualizing encounters of replication forks with ICLs as they occur in living cells. Surprisingly, the most frequent patterns were consistent with replication traverse of an ICL, without lesion repair. The traverse frequency was strongly reduced by inactivation of the translocase and DNA binding activities of the FANCM/MHF complex. The results indicate that translocase-based mechanisms enable DNA synthesis to continue past ICLs and that these lesions are not always absolute blocks to replication.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , DNA Helicases/metabolism , DNA Replication/genetics , DNA/biosynthesis , Fanconi Anemia/genetics , Tumor Suppressor Proteins/metabolism , Animals , Apoptosis Regulatory Proteins/genetics , Cricetinae , DNA Helicases/genetics , DNA-Binding Proteins , Fanconi Anemia/metabolism , Fanconi Anemia/pathology , Humans , Mice , Multiprotein Complexes/metabolism , Tumor Suppressor Proteins/genetics
5.
J Biol Chem ; 284(41): 27908-27917, 2009 Oct 09.
Article in English | MEDLINE | ID: mdl-19684342

ABSTRACT

Interstrand cross-links (ICLs) are absolute blocks to transcription and replication and can provoke genomic instability and cell death. Studies in bacteria define a two-stage repair scheme, the first involving recognition and incision on either side of the cross-link on one strand (unhooking), followed by recombinational repair or lesion bypass synthesis. The resultant monoadduct is removed in a second stage by nucleotide excision repair. In mammalian cells, there are multiple, but poorly defined, pathways, with much current attention on repair in S phase. However, many questions remain, including the efficiency of repair in the absence of replication, the factors involved in cross-link recognition, and the timing and demarcation of the first and second repair cycles. We have followed the repair of laser-localized lesions formed by psoralen (cross-links/monoadducts) and angelicin (only monoadducts) in mammalian cells. Both were repaired in G(1) phase by nucleotide excision repair-dependent pathways. Removal of psoralen adducts was blocked in XPC-deficient cells but occurred with wild type kinetics in cells deficient in DDB2 protein (XPE). XPC protein was rapidly recruited to psoralen adducts. However, accumulation of DDB2 was slow and XPC-dependent. Inhibition of repair DNA synthesis did not interfere with DDB2 recruitment to angelicin but eliminated recruitment to psoralen. Our results demonstrate an efficient ICL repair pathway in G(1) phase cells dependent on XPC, with entry of DDB2 only after repair synthesis that completes the first repair cycle. DDB2 accumulation at sites of cross-link repair is a marker for the start of the second repair cycle.


Subject(s)
Cross-Linking Reagents/pharmacology , DNA Damage , DNA Repair , DNA/drug effects , DNA/genetics , G1 Phase/genetics , Lasers , Animals , Cell Line , DNA/chemistry , DNA/metabolism , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Ficusin/pharmacology , Furocoumarins/pharmacology , Humans , Intercalating Agents/pharmacology , Molecular Structure
6.
Biochemistry ; 47(18): 5068-77, 2008 May 06.
Article in English | MEDLINE | ID: mdl-18410127

ABSTRACT

Alternate DNA structures other than double-stranded B-form DNA can potentially impede cellular processes such as transcription and replication. The DNA triplex helix and G4 tetraplex structures that form by Hoogsteen hydrogen bonding are two examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human replication protein A (RPA), a single-stranded DNA binding protein that is implicated in all facets of DNA metabolism, to destabilize DNA triplexes and tetraplexes. Biochemical studies demonstrate that RPA efficiently melts an intermolecular DNA triple helix consisting of a pyrimidine motif third strand annealed to a 4 kb duplex DNA fragment at protein concentrations equimolar to the triplex substrate. Heterologous single-stranded DNA binding proteins ( Escherichia coli SSB, T4 gene 32) melt the triplex substrate very poorly or not at all, suggesting that the triplex destabilizing effect of RPA is specific. In contrast to the robust activity on DNA triplexes, RPA does not melt intermolecular G4 tetraplex structures. Cellular assays demonstrated increased triplex DNA content when RPA is transiently repressed, suggesting that RPA melting of triple helical structures is physiologically important. On the basis of our results, we suggest that the abundance of RPA known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form from human genomic DNA sequences.


Subject(s)
DNA/chemistry , DNA/metabolism , Nucleic Acid Conformation , Replication Protein A/metabolism , Base Sequence , DNA, Single-Stranded/chemistry , HeLa Cells , Humans , Molecular Sequence Data , Replication Protein A/genetics , Temperature
7.
Biochemistry ; 46(35): 10222-33, 2007 Sep 04.
Article in English | MEDLINE | ID: mdl-17691818

ABSTRACT

We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2'-O-methylribose (2'-OMe) and 2'-O-aminoethylribose (2'-AE) residues. Active TFOs contained four contiguous 2'-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2'-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2'-AE residues, the conformationally locked BNA/LNA ribose with a 2'-O,4'-C-methylene bridge, or the 2'-O,4'-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The Tm value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences.


Subject(s)
DNA/chemistry , Oligonucleotides/chemistry , Ribose/chemistry , Animals , Base Pairing , Binding Sites , Biological Assay , Bridged-Ring Compounds/chemistry , Carbohydrates/chemistry , Cricetinae , DNA/genetics , Electroporation , Ficusin/chemistry , Gene Targeting/methods , Hypoxanthine Phosphoribosyltransferase , Models, Chemical , Nucleic Acid Conformation , Nucleic Acid Denaturation , Nucleic Acid Heteroduplexes , Oligonucleotides/pharmacology , Ribose/analogs & derivatives , Sensitivity and Specificity , Temperature
8.
Bioconjug Chem ; 18(2): 431-7, 2007.
Article in English | MEDLINE | ID: mdl-17373769

ABSTRACT

DNA interstrand cross-links are formed by chemotherapy drugs as well as by products of normal oxidative metabolism. Despite their importance, the pathways of cross-link metabolism are poorly understood. Laser confocal microscopy has become a powerful tool for studying the repair of DNA lesions that can be detected by immunofluorescent reagents. In order to apply this approach to cross-link repair, we have synthesized conjugates of 4,5',8-trimethylpsoralen (TMP) and easily detected compounds such as Lissamine rhodamine B sulfonyl chloride (LRB-SC), biotin, and digoxigenin. These conjugates are activated by UVA, and we have analyzed the intracellular localization of DNA damage and DNA reactivity by confocal and immunofluorescence microscopy. The LRB-SC-TMP conjugate 2 appeared mainly in the mitochondria, while the biotin-TMP conjugate 4 preferentially localized in the cytoplasm. Adducts formed by UVA and digoxigenin conjugates of TMP 7a and 4,5'-dimethylangelicin (DMA) 7b, which forms only monoadducts, were largely localized to the nucleus. Exposure of cells incubated with 7a and 7b to a 364 nm UV laser directed toward defined nuclear regions of interest resulted in localized adduct formation which could be visualized by immunofluorescence. Repair-proficient cells were able to remove the photoadducts, while repair-deficient cells were unable to repair the damage. The results indicated that the digoxigenin-TMP conjugate 7a and digoxigenin-DMA conjugate 7b can be used for studying the repair of laser localized DNA monoadducts and cross-links.


Subject(s)
Cross-Linking Reagents/chemistry , DNA/radiation effects , Genome , Lasers , Trioxsalen/chemistry , Animals , Biotin/chemistry , Biotin/metabolism , CHO Cells , Cricetinae , Cricetulus , Cross-Linking Reagents/metabolism , DNA/chemistry , DNA/metabolism , DNA Damage , DNA Repair , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Digoxigenin/chemistry , Digoxigenin/metabolism , Endonucleases/deficiency , Endonucleases/genetics , HeLa Cells , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Rhodamines/chemistry , Rhodamines/metabolism , Trioxsalen/metabolism , Ultraviolet Rays
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