scholarly journals A covalent protein–DNA 5′-product adduct is generated following AP lyase activity of human ALKBH1 (AlkB homologue 1)

2013 ◽  
Vol 452 (3) ◽  
pp. 509-518 ◽  
Author(s):  
Tina A. Müller ◽  
Megan M. Andrzejak ◽  
Robert P. Hausinger
Keyword(s):  
Dna Adduct ◽  
Covalent Attachment ◽  
Main Site ◽  
Chemical Mechanisms ◽  
Covalent Adduct ◽  
Lysine Residues ◽  
Lyase Activity ◽  
Ap Lyase ◽  
Covalent Intermediate

ALKBH1 (AlkB homologue 1) is a mammalian AlkB (2-oxoglutarate-dependent dioxygenase) homologue that possesses AP (abasic or apurinic/apyrimidinic) lyase activity. The AP lyase reaction is catalysed by imine formation with an active site lysine residue, and a covalent intermediate can be trapped in the presence of NaBH4. Surprisingly, ALKBH1 also forms a stable protein–DNA adduct in the absence of a reducing agent. Experiments with different substrates demonstrated that the protein covalently binds to the 5′ DNA product, i.e. the fragment containing an α,β-unsaturated aldehyde. The N-terminal domain of ALKBH1 was identified as the main site of linkage with DNA. By contrast, mutagenesis studies suggest that the primary catalytic residue forming the imine linkage is Lys133, with Lys154 and other lysine residues in this region serving in opportunistic roles. These findings confirm the classification of ALKBH1 as an AP lyase, identify the primary and a secondary lysine residues involved in the lyase reaction, and demonstrate that the protein forms a covalent adduct with the 5′ DNA product. We propose two plausible chemical mechanisms to account for the covalent attachment.

scholarly journals Reductive alkylation of ribosomes as a probe to the topography of ribosomal proteins*

1974 ◽  
Vol 143 (3) ◽  
pp. 607-612 ◽  
Author(s):  
Graham Moore ◽  
Robert R. Crichton
Keyword(s):  
Escherichia Coli ◽  
Protein Complex ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Two Dimensional ◽  
Reductive Alkylation ◽  
Lysine Residues

Escherichia coli ribosomes were treated with a number of different aldehydes of various sizes in the presence of NaBH4. After incorporation of either 3H or 14C, the ribosomal proteins were separated by two-dimensional polyacrylamide-gel electrophoresis and the extent of alkylation of the lysine residues in each protein was measured. The same pattern of alkylation was observed with the four reagents used, namely formaldehyde, acetone, benzaldehyde and 3,4,5-trimethoxybenzaldehyde. Every protein in 30S and 50S subunits was modified, although there was considerable variation in the degree of alkylation of individual proteins. A topographical classification of ribosomal proteins is presented, based on the degree of exposure of lysine residues. The data indicate that every protein of the ribosome has at least one lysine residue exposed at or near the surface of the ribonucleo-protein complex.

scholarly journals Development of a yeast model to study the contribution of vacuolar polyphosphate metabolism to lysine polyphosphorylation

2019 ◽  
Vol 295 (6) ◽  
pp. 1439-1451 ◽  
Author(s):  
Cristina Azevedo ◽  
Yann Desfougères ◽  
Yannasittha Jiramongkol ◽  
Hamish Partington ◽  
Sasanan Trakansuebkul ◽  
...  
Keyword(s):  
Cell Lysis ◽  
Covalent Attachment ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Target Domain ◽  
Inorganic Polyphosphate ◽  
Topoisomerase 1 ◽  
Lysine Residues ◽  
Polyphosphate Metabolism ◽  
Nuclear Targets

A recently-discovered protein post-translational modification, lysine polyphosphorylation (K-PPn), consists of the covalent attachment of inorganic polyphosphate (polyP) to lysine residues. The nonenzymatic nature of K-PPn means that the degree of this modification depends on both polyP abundance and the amino acids surrounding the modified lysine. K-PPn was originally discovered in budding yeast (Saccharomyces cerevisiae), in which polyP anabolism and catabolism are well-characterized. However, yeast vacuoles accumulate large amounts of polyP, and upon cell lysis, the release of the vacuolar polyP could nonphysiologically cause K-PPn of nuclear and cytosolic targets. Moreover, yeast vacuoles possess two very active endopolyphosphatases, Ppn1 and Ppn2, that could have opposing effects on the extent of K-PPn. Here, we characterized the contribution of vacuolar polyP metabolism to K-PPn of two yeast proteins, Top1 (DNA topoisomerase 1) and Nsr1 (nuclear signal recognition 1). We discovered that whereas Top1-targeting K-PPn is only marginally affected by vacuolar polyP metabolism, Nsr1-targeting K-PPn is highly sensitive to the release of polyP and of endopolyphosphatases from the vacuole. Therefore, to better study K-PPn of cytosolic and nuclear targets, we constructed a yeast strain devoid of vacuolar polyP by targeting the exopolyphosphatase Ppx1 to the vacuole and concomitantly depleting the two endopolyphosphatases (ppn1Δppn2Δ, vt-Ppx1). This strain enabled us to study K-PPn of cytosolic and nuclear targets without the interfering effects of cell lysis on vacuole polyP and of endopolyphosphatases. Furthermore, we also define the fundamental nature of the acidic amino acid residues to the K-PPn target domain.

Adenine Glycosylase MutY of Corynebacterium pseudotuberculosis presents the antimutator phenotype and evidences of glycosylase/AP lyase activity in vitro

2016 ◽  
Vol 44 ◽  
pp. 318-329 ◽  
Author(s):  
Rafael Cançado de Faria ◽  
Liliane Gonçalves Vila-Nova ◽  
Mainá Bitar ◽  
Bruno Carvalho Resende ◽  
Larissa Sousa Arantes ◽  
...  
Keyword(s):  
Corynebacterium Pseudotuberculosis ◽  
Lyase Activity ◽  
Ap Lyase ◽  
Adenine Glycosylase

scholarly journals A non-canonical role for the DNA glycosylase NEIL3 in suppressing APE1 endonuclease-mediated ssDNA damage

2020 ◽  
Vol 295 (41) ◽  
pp. 14222-14235 ◽  
Author(s):  
Anh Ha ◽  
Yunfeng Lin ◽  
Shan Yan
Keyword(s):  
Excision Repair ◽  
Dna Glycosylase ◽  
Genome Integrity ◽  
Ap Endonuclease ◽  
C Terminus ◽  
Egg Extracts ◽  
Lyase Activity ◽  
Repair Pathways ◽  
Base Excision ◽  
Ap Lyase

The DNA glycosylase NEIL3 has been implicated in DNA repair pathways including the base excision repair and the interstrand cross-link repair pathways via its DNA glycosylase and/or AP lyase activity, which are considered canonical roles of NEIL3 in genome integrity. Compared with the other DNA glycosylases NEIL1 and NEIL2, Xenopus laevis NEIL3 C terminus has two highly conserved zinc finger motifs containing GRXF residues (designated as Zf-GRF). It has been demonstrated that the minor AP endonuclease APE2 contains only one Zf-GRF motif mediating interaction with single-strand DNA (ssDNA), whereas the major AP endonuclease APE1 does not. It appears that the two NEIL3 Zf-GRF motifs (designated as Zf-GRF repeat) are dispensable for its DNA glycosylase and AP lyase activity; however, the potential function of the NEIL3 Zf-GRF repeat in genome integrity remains unknown. Here, we demonstrate evidence that the NEIL3 Zf-GRF repeat was associated with a higher affinity for shorter ssDNA than one single Zf-GRF motif. Notably, our protein–protein interaction assays show that the NEIL3 Zf-GRF repeat but not one Zf-GRF motif interacted with APE1 but not APE2. We further reveal that APE1 endonuclease activity on ssDNA but not on dsDNA is compromised by a NEIL3 Zf-GRF repeat, whereas one Zf-GRF motif within NEIL3 is not sufficient to prevent such activity of APE1. In addition, COMET assays show that excess NEIL3 Zf-GRF repeat reduces DNA damage in oxidative stress in Xenopus egg extracts. Together, our results suggest a noncanonical role of NEIL3 in genome integrity via its distinct Zf-GRF repeat in suppressing APE1 endonuclease-mediated ssDNA breakage.

The new world of inorganic polyphosphates

2016 ◽  
Vol 44 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Cristina Azevedo ◽  
Adolfo Saiardi
Keyword(s):  
Protein Modification ◽  
Biochemical Characterization ◽  
Covalent Attachment ◽  
Detection Methods ◽  
Inorganic Polyphosphate ◽  
Post Translational Modifications ◽  
Topoisomerase 1 ◽  
Lysine Residues ◽  
Acidic Conditions

Post-translational modifications (PTMs) add regulatory features to proteins that help establish the complex functional networks that make up higher organisms. Advances in analytical detection methods have led to the identification of more than 200 types of PTMs. However, some modifications are unstable under the present detection methods, anticipating the existence of further modifications and a much more complex map of PTMs. An example is the recently discovered protein modification polyphosphorylation. Polyphosphorylation is mediated by inorganic polyphosphate (polyP) and represents the covalent attachment of this linear polymer of orthophosphate to lysine residues in target proteins. This modification has eluded MS analysis as both polyP itself and the phosphoramidate bonds created upon its reaction with lysine residues are highly unstable in acidic conditions. Polyphosphorylation detection was only possible through extensive biochemical characterization. Two targets have been identified: nuclear signal recognition 1 (Nsr1) and its interacting partner, topoisomerase 1 (Top1). Polyphosphorylation occurs within a conserved N-terminal polyacidic serine (S) and lysine (K) rich (PASK) cluster. It negatively regulates Nsr1–Top1 interaction and impairs Top1 enzymatic activity, namely relaxing supercoiled DNA. Modulation of cellular levels of polyP regulates Top1 activity by modifying its polyphosphorylation status. Here we discuss the significance of the recently identified new role of inorganic polyP.

scholarly journals Post-translational regulation of ubiquitin signaling

2019 ◽  
Vol 218 (6) ◽  
pp. 1776-1786 ◽  
Author(s):  
Lei Song ◽  
Zhao-Qing Luo
Keyword(s):  
Translational Regulation ◽  
Covalent Attachment ◽  
Immune Disorders ◽  
Post Translational Modification ◽  
Polyubiquitin Chains ◽  
Cellular Processes ◽  
Lysine Residues ◽  
Substrate Proteins ◽  
Ubiquitination Machinery ◽  
Integrate Development

Ubiquitination regulates many essential cellular processes in eukaryotes. This post-translational modification (PTM) is typically achieved by E1, E2, and E3 enzymes that sequentially catalyze activation, conjugation, and ligation reactions, respectively, leading to covalent attachment of ubiquitin, usually to lysine residues of substrate proteins. Ubiquitin can also be successively linked to one of the seven lysine residues on ubiquitin to form distinctive forms of polyubiquitin chains, which, depending upon the lysine used and the length of the chains, dictate the fate of substrate proteins. Recent discoveries revealed that this ubiquitin code is further expanded by PTMs such as phosphorylation, acetylation, deamidation, and ADP-ribosylation, on ubiquitin, components of the ubiquitination machinery, or both. These PTMs provide additional regulatory nodes to integrate development or insulting signals with cellular homeostasis. Understanding the precise roles of these PTMs in the regulation of ubiquitin signaling will provide new insights into the mechanisms and treatment of various human diseases linked to ubiquitination, including neurodegenerative diseases, cancer, infection, and immune disorders.

scholarly journals Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance inEscherichia colimutant

2009 ◽  
Vol 14 (2) ◽  
pp. 261-270 ◽  
Author(s):  
Masashi Takao ◽  
Yoshitsugu Oohata ◽  
Kengo Kitadokoro ◽  
Kumiko Kobayashi ◽  
Shigenori Iwai ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Resistance ◽  
Single Stranded Dna ◽  
Oxidative Stress Resistance ◽  
Lyase Activity ◽  
Ap Lyase
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