Fast, inexpensive, high-yielding, site-selective, chemical synthesis of cross-linked DNA duplexes via hydrazone formation between N[superscript 4]-aminocytidine and Ap-sites

2017 ◽  
Author(s):  
◽  
Jacqueline Gamboa Varela
Keyword(s):  
High Yield ◽  
Dna Duplexes ◽  
Cross Link ◽  
University Of Missouri ◽  
Cellular Processes ◽  
Abasic Sites ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross ◽  
High Yields

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] DNA is the central molecule of biology as it stores the genetic information for cells to properly function and develop. Modifications to the DNA can stall cellular processes such as replication and transcription, leading the cell to recruit repair machinery or in some cases undergo apoptosis. Interstrand cross-links are particularly significant types of DNA damage because they prevent strand separation required for replication and transcription. Cross-links involve bonding between the two strands of DNA. The rate and mechanism of cross-link repair in cells are not well understood. A significant challenge in the study of cross-link repair is the synthesis of chemically well-defined DNA cross-links. Here we summarize the preparation of cross-links derived from the hydrazone formation between a non-natural nucleobase N4-aminocytidine and abasic sites in duplex DNA. The cross-link was generated rapidly and in high yield. The cross-link is stable under physiological conditions but, interestingly, can be reversibly dissociated and re-formed by thermal cycling between 20-80 [degrees]C. We provided evidence that the cross-link is stable against multiple agents and the cross-link is reversible. We used this chemistry to prepare structurally diverse cross-links for the utilization in cross-link repair studies. Overall, we developed a synthetic cross-link that is easily and rapidly prepared from commercially available reagents in high yields, at defined locations in duplexed DNA.

scholarly journals Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand.

1997 ◽  
Vol 17 (12) ◽  
pp. 6822-6830 ◽  
Author(s):  
T Bessho ◽  
D Mu ◽  
A Sancar
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Repair System ◽  
Cross Link ◽  
Unique Challenge ◽  
Cell Extracts ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.

FANCD2 Localizes to Cross-Linked Induced Nuclear Foci That Are Distinct From Those to Which αaII Spectrin and the Cross-Link Repair Protein, XPF, Co-Localize, Indicating An Involvement of These Proteins in Different Steps of the DNA Interstrand Cross-Link Repair Process.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3196-3196
Author(s):  
Pan Zhang ◽  
Deepa Sridharan ◽  
Michael Acosta ◽  
Muriel Lambert
Keyword(s):  
Time Course ◽  
Repair Process ◽  
Myelogenous Leukemia ◽  
Cross Linking ◽  
Cross Link ◽  
Repair Protein ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Nuclear Foci ◽  
The Cross

Abstract Abstract 3196 Poster Board III-133 The hereditary bone marrow failure disorder, Fanconi anemia (FA), is characterized by a markedly increased incidence of acute myelogenous leukemia, diverse congenital abnormalities and a defect in ability to repair DNA interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. One of the FA proteins which is thought to play an important role in the repair of DNA interstrand cross-links is FANCD2, which is known to form nuclear foci after cross-link damage. The present study was undertaken in order to get a better understanding of the relationship between aSpII and FANCD2, whether they interact with each other during the DNA repair process and co-localize in damage-induced nuclear foci. Immunofluorescence microscopy was carried out to determine whether these proteins co-localized in nuclear foci after cells were damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP) or mitomycin C (MMC). Time course measurements showed that FANCD2 foci were first visible at 2 hours after damage and increased up to 16 hours and were still present at 72 hours after damage. This time course of foci formation correlated with levels of monoubiquitination of FANCD2. Measurement of gH2AX foci formation showed that the time course of foci formation was similar to that of FANCD2 measured up to 72 hours post damage. In contrast, aSpII foci were first visible between 8-10 hours after damage. The number of these foci peaked at 16 hours and by 24 hours foci were no longer observed. Co-localization studies showed that there was little co-localization of the FANCD2 and aSpII foci over this time course. This indicates that these two proteins may be involved in different steps in the DNA interstrand cross-link repair process. Based on models that have been proposed for the role of FANCD2 in the repair of DNA interstrand cross-links, we propose that, after DNA damage, FANCD2 localizes at DNA replication forks stalled at sites of interstrand cross-links and aids in the assembly of proteins at this site. This is followed by localization of aSpII and XPF and other proteins involved in the initial incision steps in DNA interstrand cross-link repair where they play a role in the unhooking of the cross-link. FANCD2 is then involved in subsequent steps in the repair process, which involve homologous recombination. Thus two proteins, FANCD2 and aSpII, both of which have been shown to be critical for the DNA interstrand cross-link repair process may be involved in different or distinct steps in this repair process. Deficiencies in these proteins would impact on DNA interstrand cross-link repair and, as we have shown for aIISp, would have an adverse effect on the genomic stability of FA cells. . Disclosures No relevant conflicts of interest to declare.

scholarly journals Processing of a Psoralen DNA Interstrand Cross-link by XPF-ERCC1 Complex in Vitro

2007 ◽  
Vol 283 (3) ◽  
pp. 1275-1281 ◽  
Author(s):  
Laura A. Fisher ◽  
Mika Bessho ◽  
Tadayoshi Bessho
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Dependent Manner ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross ◽  
Stalled Fork

The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5′ to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICL-specific incision on the 3′-side of an ICL. The ICL-specific 3′-incision, along with the 5′-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork.

scholarly journals DNA Interstrand Cross-Links Induce Futile Repair Synthesis in Mammalian Cell Extracts

2000 ◽  
Vol 20 (7) ◽  
pp. 2446-2454 ◽  
Author(s):  
David Mu ◽  
Tadayoshi Bessho ◽  
Lubomir V. Nechev ◽  
David J. Chen ◽  
Thomas M. Harris ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Excision Repair ◽  
Mutant Cell ◽  
Complex Structure ◽  
Cross Link ◽  
Repair Synthesis ◽  
Cell Extracts ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross

ABSTRACT DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5′ to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3′-to-5′ exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.

scholarly journals Chemical and strutural properties of DNA-abasic site cross-links

2014 ◽  
Author(s):  
◽  
Nathan Price
Keyword(s):  
Gel Electrophoresis ◽  
Structural Information ◽  
Sequence Specificity ◽  
Abasic Site ◽  
Cross Link ◽  
Dna Sequence Specificity ◽  
Abasic Sites ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Synthetic Standard

Here, we report on the formation of DNA interstrand cross-links. These cross-links form from abasic sites, an endogenous type of DNA damage. We have utilized gel electrophoresis, NMR, mass spectrometry and liquid chromatography to identify the location, DNA sequence specificity, stability and some structural information for these interstrand cross-links. Additionally, we have synthesized a small molecule standard which is identical to the enzymatically digested cross-link from duplex DNA. This synthetic standard can be used to increase sensitivity for future detection of DNA-abasic site cross-links.

scholarly journals DNA Polymerase II (polB) Is Involved in a New DNA Repair Pathway for DNA Interstrand Cross-Links inEscherichia coli

1999 ◽  
Vol 181 (9) ◽  
pp. 2878-2882 ◽  
Author(s):  
Mark Berardini ◽  
Patricia L. Foster ◽  
Edward L. Loechler
Keyword(s):  
Dna Polymerase ◽  
Nitrogen Mustard ◽  
Wild Type ◽  
Cross Link ◽  
Replication Efficiency ◽  
E Coli ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross ◽  
Dna Polymerase Ii

ABSTRACT DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be ∼0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in apolB + (∼0.6) than in a ΔpolB(∼0.1) strain. Complementation with apolB +-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type <polB < recA < polB recA ∼ uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing,E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.

An approach for the synthesis of duplexes containing N3T-butyl-N3T interstrand cross-links via a bisphosphoramidite strategy

2007 ◽  
Vol 85 (4) ◽  
pp. 249-256 ◽  
Author(s):  
Christopher James Wilds ◽  
Ernest Palus ◽  
Anne Marietta Noronha
Keyword(s):  
Dna Repair ◽  
Solid Phase ◽  
Dna Duplexes ◽  
Cross Link ◽  
Phase Synthesis ◽  
Chemically Modified ◽  
Biophysical Studies ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Thymidine Dimer

DNA duplexes containing an interstrand cross-link have been synthesized utilizing a bis-3′-O-phosphoramidite deoxythymidine dimer where the N3 atoms are bridged by a butyl linker. With this approach sufficient quantities of high purity cross-linked duplexes are obtained that will enable various biochemical and structural studies to aid in research directed towards understanding the mechanism of interstrand cross-linked DNA repair. This methodology has advantages over a previously reported method to synthesize cross-linked DNA duplexes involving a monophosphoramidite of the same cross-linked thymidine dimer including circumventing the use of costly 5′-O-deoxyphosphoramidites in the assembly of the cross-linked duplex by solid-phase synthesis. This strategy can be employed to produce cross-linked duplexes in which the lesions are engineered to have a directly opposed (1–1) or staggered (1–2 or 2–1) orientations. Biophysical studies of duplexes containing this N3T-butyl-N3T cross-link in staggered 1–2 and 2–1 orientations reveal that both duplexes have a higher Tm than a non-cross-linked duplex suggesting that these linkages do not result in the destabilization of duplex DNA. Circular dichroism spectra of the 1–2 and 2–1 cross-linked duplexes exhibit minor differences from B-form structure, which correlates with molecular modeling studies.Key words: chemically modified oligonucleotides, interstrand cross-link, DNA adduct, DNA repair.

scholarly journals Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
Author(s):  
E J Aamodt ◽  
J G Culotti
Keyword(s):  
Caenorhabditis Elegans ◽  
Apparent Molecular Weight ◽  
Cross Link ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Associated Proteins ◽  
Cross Links ◽  
The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

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