Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
J Am Chem Soc ; 144(23): 10471-10482, 2022 06 15.
Article in English | MEDLINE | ID: mdl-35612610

ABSTRACT

The hydrolytic loss of coding bases from cellular DNA is a common and unavoidable reaction. The resulting abasic sites can undergo ß-elimination of the 3'-phosphoryl group to generate a strand break with an electrophilic α,ß-unsaturated aldehyde residue on the 3'-terminus. The work reported here provides evidence that the thiol residue of the cellular tripeptide glutathione rapidly adds to the alkenal group on the 3'-terminus of an AP-derived strand break. The resulting glutathionylated adduct is the only major cleavage product observed when ß-elimination occurs at an AP site in the presence of glutathione. Formation of the glutathionylated cleavage product is reversible, but in the presence of physiological concentrations of glutathione, the adduct persists for days. Biochemical experiments provided evidence that the 3'-phosphodiesterase activity of the enzyme apurinic/apyrimidinic endonuclease (APE1) can remove the glutathionylated sugar remnant from an AP-derived strand break to generate the 3'OH residue required for repair via base excision or single-strand break repair pathways. The results suggest that a previously unrecognized 3'glutathionylated sugar remnant─and not the canonical α,ß-unsaturated aldehyde end group─may be the true strand cleavage product arising from ß-elimination at an abasic site in cellular DNA. This work introduces the 3'glutathionylated cleavage product as the major blocking group that must be trimmed to enable repair of abasic site-derived strand breaks by the base excision repair or single-strand break repair pathways.


Subject(s)
DNA Damage , DNA Repair , Aldehydes , DNA/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Glutathione , Sugars
2.
Chem Res Toxicol ; 35(2): 203-217, 2022 02 21.
Article in English | MEDLINE | ID: mdl-35124963

ABSTRACT

Abasic sites are common in cellular and synthetic DNA. As a result, it is important to characterize the chemical fate of these lesions. Amine-catalyzed strand cleavage at abasic sites in DNA is an important process in which conversion of small amounts of the ring-opened abasic aldehyde residue to an iminium ion facilitates ß-elimination of the 3'-phosphoryl group. This reaction generates a trans-α,ß-unsaturated iminium ion on the 3'-terminus of the strand break as an obligate intermediate. The canonical product expected from amine-catalyzed cleavage at an AP site is the corresponding trans-α,ß-unsaturated aldehyde sugar remnant resulting from hydrolysis of this iminium ion. Interestingly, a handful of studies have reported noncanonical 3'-sugar remnants generated by amine-catalyzed strand cleavage, but the formation and properties of these products are not well-understood. To address this knowledge gap, a nucleoside system was developed that enabled chemical characterization of the sugar remnants generated by amine-catalyzed ß-elimination in the 2-deoxyribose system. The results predict that amine-catalyzed strand cleavage at an AP site under physiological conditions has the potential to reversibly generate noncanonical cleavage products including cis-alkenal, 3-thio-2,3-dideoxyribose, and 2-deoxyribose groups alongside the canonical trans-alkenal residue on the 3'-terminus of the strand break. Thus, the model reactions provide evidence that the products generated by amine-catalyzed strand cleavage at abasic sites in cellular DNA may be more complex that commonly thought, with trans-α,ß-unsaturated iminium ion intermediates residing at the hub of interconverting product mixtures. The results expand the list of possible 3'-sugar remnants arising from amine-catalyzed cleavage of abasic sites in DNA that must be chemically or enzymatically removed for the completion of base excision repair and single-strand break repair in cells.


Subject(s)
Amines/chemistry , Biomimetic Materials/chemistry , DNA/drug effects , Deoxyribose/chemistry , Nucleosides/chemistry , Catalysis , DNA Damage , DNA Repair , Nucleic Acid Conformation
3.
Chem Res Toxicol ; 35(2): 218-232, 2022 02 21.
Article in English | MEDLINE | ID: mdl-35129338

ABSTRACT

Hydrolytic loss of nucleobases from the deoxyribose backbone of DNA is one of the most common unavoidable types of damage in synthetic and cellular DNA. The reaction generates abasic sites in DNA, and it is important to understand the properties of these lesions. The acidic nature of the α-protons of the ring-opened abasic aldehyde residue facilitates the ß-elimination of the 3'-phosphoryl group. This reaction is expected to generate a DNA strand break with a phosphoryl group on the 5'-terminus and a trans-α,ß-unsaturated aldehyde residue on the 3'-terminus; however, a handful of studies have identified noncanonical sugar remnants on the 3'-terminus, suggesting that the products arising from strand cleavage at apurinic/apyrimidinic sites in DNA may be more complex than commonly thought. We characterized the strand cleavage induced by the treatment of an abasic site-containing DNA oligonucleotide with heat, NaOH, piperidine, spermine, and the base excision repair glycosylases Fpg and Endo III. The results showed that under multiple conditions, cleavage at an abasic site in a DNA oligomer generated noncanonical sugar remnants including cis-α,ß-unsaturated aldehyde, 2-deoxyribose, and 3-thio-2,3-dideoxyribose products on the 3'-terminus of the strand break.


Subject(s)
Amines/pharmacology , DNA Glycosylases/metabolism , DNA/drug effects , DNA/metabolism , Hot Temperature , Sodium Hydroxide/pharmacology , Amines/chemistry , DNA Cleavage , DNA Repair , Sodium Hydroxide/chemistry
4.
J Am Chem Soc ; 143(37): 15344-15357, 2021 09 22.
Article in English | MEDLINE | ID: mdl-34516735

ABSTRACT

Interstrand DNA cross-links (ICLs) are cytotoxic because they block the strand separation required for read-out and replication of the genetic information in duplex DNA. The unavoidable formation of ICLs in cellular DNA may contribute to aging, neurodegeneration, and cancer. Here, we describe the formation and properties of a structurally complex ICL derived from an apurinic/apyrimidinic (AP) site, which is one of the most common endogenous lesions in cellular DNA. The results characterize a cross-link arising from aza-Michael addition of the N2-amino group of a guanine residue to the electrophilic sugar remnant generated by spermine-mediated strand cleavage at an AP site in duplex DNA. An α,ß-unsaturated iminium ion is the critical intermediate involved in ICL formation. Studies employing the bacteriophage φ29 polymerase provided evidence that this ICL can block critical DNA transactions that require strand separation. The results of biochemical studies suggest that this complex strand break/ICL might be repaired by a simple mechanism in which the 3'-exonuclease action of the enzyme apurinic/apyrimidinic endonuclease (APE1) unhooks the cross-link to initiate repair via the single-strand break repair pathway.


Subject(s)
DNA Damage , DNA Repair , DNA/chemistry , Nucleic Acid Conformation
5.
DNA Repair (Amst) ; 98: 103029, 2021 02.
Article in English | MEDLINE | ID: mdl-33385969

ABSTRACT

Genome integrity is essential for life and, as a result, DNA repair systems evolved to remove unavoidable DNA lesions from cellular DNA. Many forms of life possess the capacity to remove interstrand DNA cross-links (ICLs) from their genome but the identity of the naturally-occurring, endogenous substrates that drove the evolution and retention of these DNA repair systems across a wide range of life forms remains uncertain. In this review, we describe more than a dozen chemical processes by which endogenous ICLs plausibly can be introduced into cellular DNA. The majority involve DNA degradation processes that introduce aldehyde residues into the double helix or reactions of DNA with endogenous low molecular weight aldehyde metabolites. A smaller number of the cross-linking processes involve reactions of DNA radicals generated by oxidation.


Subject(s)
DNA Adducts/metabolism , DNA Repair , Animals , Humans
6.
J Biol Chem ; 295(46): 15566-15575, 2020 11 13.
Article in English | MEDLINE | ID: mdl-32878989

ABSTRACT

The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows that the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA-processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.


Subject(s)
DNA, Single-Stranded/metabolism , N-Glycosyl Hydrolases/metabolism , Amino Acid Sequence , Animals , Crystallography, X-Ray , DNA/metabolism , DNA Replication , DNA, Single-Stranded/chemistry , Humans , Mice , N-Glycosyl Hydrolases/antagonists & inhibitors , N-Glycosyl Hydrolases/genetics , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Zinc Fingers
7.
DNA Repair (Amst) ; 86: 102752, 2020 02.
Article in English | MEDLINE | ID: mdl-31923807

ABSTRACT

Interstrand DNA-DNA cross-links (ICLs) are generated by endogenous processes, drugs, and environmental toxins. Understanding the cellular pathways by which various ICLs are repaired is critical to understanding their biological effects. Recent studies showed that replication-dependent repair of an ICL derived from the reaction of an abasic (AP) site with an adenine residue (dA) on the opposing strand of duplex DNA proceeds via a novel mechanism in which the DNA glycosylase NEIL3 unhooks the ICL. Here we examined the ability of the glycosylase domain of murine NEIL3 (MmuNEIL3-GD) to unhook dA-AP ICLs. The enzyme selectively unhooks the dA-AP ICL located at the duplex/single-strand junction of splayed duplexes that model the strand-separated DNA at the leading edge of a replication fork. We show that the ability to unhook the dA-AP ICL is a specialized function of NEIL3 as this activity is not observed in other BER enzymes. Importantly, NEIL3 only unhooks the dA-AP ICL when the AP residue is located on what would be the leading template strand of a model replication fork. The same specificity for the leading template strand was observed with a 5,6-dihydrothymine monoadduct, demonstrating that this preference is a general feature of the glycosylase and independent of the type of DNA damage. Overall, the results show that the glycosylase domain of NEIL3, lacking the C-terminal NPL4 and GRF zinc finger motifs, is competent to unhook the dA-AP ICL in splayed substrates and independently enforces important substrate preferences on the repair process.


Subject(s)
DNA/chemistry , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Animals , Cross-Linking Reagents , Mice , Nucleic Acid Conformation , Protein Domains , Thymine/analogs & derivatives , Thymine/chemistry , Thymine/metabolism
8.
ACS Chem Biol ; 14(7): 1481-1489, 2019 07 19.
Article in English | MEDLINE | ID: mdl-31259519

ABSTRACT

Efficient methods for the site-specific installation of structurally defined interstrand cross-links in duplex DNA may be useful in a wide variety of fields. The work described here developed a high-yield synthesis of chemically stable interstrand cross-links resulting from a reductive amination reaction between an abasic site and the noncanonical nucleobase 2-aminopurine in duplex DNA. Results from footprinting, liquid chromatography-mass spectrometry, and stability studies support the formation of an N2-alkylamine attachment between the 2-aminopurine residue and the Ap site. The reaction performs best when the 2-aminopurine residue on the opposing strand is offset 1 nt to the 5'-side of the abasic site. The cross-link confers substantial resistance to thermal denaturation (melting). The cross-linking reaction is fast (complete in 4 h), employs only commercially available reagents, and can be used to generate cross-linked duplexes in sufficient quantities for biophysical, structural, and DNA repair studies.


Subject(s)
2-Aminopurine/chemistry , Cross-Linking Reagents/chemistry , DNA/chemistry , Amination , Models, Molecular , Nucleic Acid Conformation , Nucleic Acid Denaturation , Oxidation-Reduction
SELECTION OF CITATIONS
SEARCH DETAIL
...