Search

Over 70,000 DNA lesions occur in the cell every day, and the inability to properly repair them can lead to mutations and destabilize the genome, resulting in carcinogenesis. The base excision repair (BER) pathway is critical for maintaining genomic integrity by repairing small base lesions, abasic sites and single‐stranded breaks. Monofunctional and bifunctional glycosylases initiate the first step of BER by recognizing and excising specific base lesions, followed by DNA end processing, gap filling, and finally nick sealing. The Nei‐like 2 (NEIL2) enzyme is a critical bifunctional DNA glycosylase in BER that preferentially excises cytosine oxidation products and abasic sites from single‐stranded, double‐stranded, and bubble‐structured DNA. NEIL2 has been implicated to have important roles in several cellular functions, including genome maintenance, participation in active demethylation, and modulation of the immune response. Several germline and somatic variants of NEIL2 with altered expression and enzymatic activity have been reported in the literature linking them to cancers. In this review, we provide an overview of NEIL2 cellular functions and summarize current findings on NEIL2 variants and their relationship to cancer. [ABSTRACT FROM AUTHOR]

ABSTRACT Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3′-to-5′ helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr’s ATPase/helicase activities and its tetrameric quaternary structure. The lhr–nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen–UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr–nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen–UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen–UVA.

Endonuclease VIII-like 3 (NEIL3) is a versatile DNA glycosylase that repairs a diverse array of chemical modifications to DNA. Unlike other glycosylases, NEIL3 has a preference for lesions within single-strand DNA and at single/double-strand DNA junctions. Beyond its canonical role in base excision repair of oxidized DNA, NEIL3 initiates replication-dependent interstrand DNA crosslink repair as an alternative to the Fanconi Anemia pathway. This review outlines our current understanding of NEIL3's biological functions, role in disease, and three-dimensional structure as it pertains to substrate specificity and catalytic mechanism. • NEIL3 expression increases in S-phase and plateaus in G2. • Expression increased in many cancers and is linked to neurodegenerative disorders. • NEIL3 excises oxidized pyrimidines and unhooks interstrand DNA crosslinks. • NEIL3 is specific for lesions in ssDNA and at ss/dsDNA junctions. • Structure lacks DNA intercalating residues present in other Nei family members. [ABSTRACT FROM AUTHOR]

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP β-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆ nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆ lhr cells by mutant lhr alleles depended on Lhr's ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA., Competing Interests: The authors declare no conflict of interest.

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ∼ 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replication-associated repair., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

[Display omitted] • Tb3+/G-quadruplex fluorescence detection of oxidative DNA damage for the first time; • Lowest detection limit due to introduction of human 8-oxoguanine DNA glycosylase for damage amplification. Development of simple, sensitive and rapid sensing strategy for DNA damage induced by chemical reagents is urgent demand. In this assay, a Tb3+/G-quadruplex fluorescence system was proposed for the sensitive detection of DNA damage induced by Fenton reaction. The specific guanine-rich oligonucleotides can form G-quadruplex induced by Tb3+, leading to an enhancement of Tb3+ emission due to energy transfer from guanine to Tb3+. After a DNA damage reaction with Fenton reagents, an obvious fluorescence decrease was observed due to the structure change of Tb3+/G-quadruplex. which was also confirmed by polyacrylamide gel electrophoresis and circular dichroism. The Fe2+-mediated Fenton reaction with as low as 0.01 μM Fe2+ and 0.04 μM H 2 O 2 induced oxidative DNA damage can be detected. Additionally, the human 8-oxoguanine DNA glycosylase (hOGG 1) was introduced to amplify DNA oxidation damage, by converting DNA oxidative nucleobases to strand break. Over 30-fold lower detection limit of 0.3 nM Fe2+ and 1.2 nM H 2 O 2 was achieved. Alternative specific DNA damage or other DNA repair enzymes produced much less fluorescence decrease, demonstrating its good selectivity. It shows the potential to become a universal screening tool for the rapid and sensitive assessment of the genotoxicity of the overwhelming number of chemicals in environment. [ABSTRACT FROM AUTHOR]

DNA-modifying enzymes act as critical regulators in a wide range of genetic functions (e.g., DNA damage & repair, DNA replication), and their aberrant expression may interfere with regular genetic functions and induce various malignant diseases including cancers. DNA-modifying enzymes have emerged as the potential biomarkers in early diagnosis of diseases and new therapeutic targets in genomic research. Consequently, the development of highly specific and sensitive biosensors for the detection of DNA-modifying enzymes is of great importance for basic biomedical research, disease diagnosis, and drug discovery. Single-molecule fluorescence detection has been widely implemented in the field of molecular diagnosis due to its simplicity, high sensitivity, visualization capability, and low sample consumption. In this paper, we summarize the recent advances in single-molecule counting-based biosensors for DNA-modifying enzyme (i.e, alkaline phosphatase, DNA methyltransferase, DNA glycosylase, flap endonuclease 1, and telomerase) assays in the past four years (2019 − 2023). We highlight the principles and applications of these biosensors, and give new insight into the future challenges and perspectives in the development of single-molecule counting-based biosensors. [Display omitted] • We review single-molecule counting-based biosensors for DNA-modifying enzyme detection. • The principles and applications of these biosensors are highlighted. • The performances of different biosensors are compared. • We discuss the future challenges and perspectives in this field. [ABSTRACT FROM AUTHOR]

DNA glycosylase assumes a pivotal role in the preservation of genome integrity by recognizing and excising damaged bases. Herein, we develop a single primer/self-template-powered cascade rolling circle amplification (RCA) and apurinic/apyrimidinic endonuclease (APE1)-mediated signal amplification for simultaneous detection of human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG). The uniqueness of this approach lies in the use of custom-designed hairpin probes to establish a prerequisite for RCA: the conformational change of hairpin probe to circular template, which effectively suppress undesired non-specific amplification. We design two hairpin probes featuring elongated stem structure and 3′-phosphate (3′-PO 4) modifications. The stems of hairpin probes are modified with glycosylase substrates and BER process can cleave hairpin probes, resulting in the generation of single-stranded DNAs (ssDNAs) with 3′-hydroxyl (3′-OH) ends. These ssDNAs can function as padlock probes to form circular template upon ligation by ligase. Ultimately, the self-template-initiated RCA and APE1-mediated signal amplification cascade occurs, generating a substantial number of free Cy3 and Cy5 fluorophores that can be simply visualized by single-molecule fluorescence detection. This method demonstrates excellent specificity and sensitivity, with a limit of detection (LOD) of 3.42 × 10-12 U/μL for hAAG and 2.41 × 10-12 U/μL for UDG. This method also can be used for screening of potential inhibitor and kinetic parameter measurement. Most importantly, this method enables accurate profiling of glycosylases expression levels in various cancer cell lines and detection of glycosylase activities at the single-cell level, offering a bright future in cancer detection, medical research, and precision medicine. • Simultaneous detection of multiple glycosylases at ultra-low concentrations. • Conformation-switchable hairpin probes and self-templated amplification reduce non-specific amplification. • One primer probe can simultaneously trigger two isothermal amplifications. • Accurate assessment of DNA glycosylase expression in different cancer cell lines. [ABSTRACT FROM AUTHOR]

DNA is the major target of radiation therapy of malignant tumors. Ionizing radiation (IR) induces a variety of DNA lesions, including chemically modified bases and strand breaks. The use of proton beam therapy for cancer treatment is ramping up, as it is expected to reduce normal tissue damage. Thus, it is important to understand the molecular mechanisms of recognition, signaling, and repair of DNA damage induced by protons in the perspective of assessing not only the risk associated with human exposure to IR but also the possibility to improve the efficacy of therapy. Here, we used targeted irradiation of nuclear regions of living cells with controlled number of protons at a high spatio-temporal resolution to detect the induced base lesions and characterize the recruitment kinetics of the specific DNA glycosylases to DNA damage sites. We show that localized irradiation with 4 MeV protons induces, in addition to DNA double strand breaks (DSBs), the oxidized bases 7,8-dihydro-8-oxoguanine (8-oxoG) and thymine glycol (TG) at the site of irradiation. Consistently, the DNA glycosylases OGG1 and NTH1, capable of excising 8-oxoG and TG, respectively, and initiating the base excision repair (BER) pathway, are recruited to the site of damage. To our knowledge, this is the first direct evidence indicating that proton microbeams induce oxidative base damage, and thus implicating BER in the repair of DNA lesions induced by protons. [Display omitted] • Proton microbeam induces, besides DNA double strand breaks (DSB), oxidative base lesions. • DNA glycosylases OGG1 and NTH1, together with DSB repair proteins, are recruited to localized proton-irradiation sites. • The localization of OGG1 and NTH1 is restricted to the area of damaged DNA, unlike γH2AX. • The OGG1 and NTH1 release from damaged DNA depends on their enzymatic activity. [ABSTRACT FROM AUTHOR]