scholarly journals AP-endonuclease 1 sculpts DNA through an anchoring tyrosine residue on the DNA intercalating loop

2020 ◽  
Author(s):  
Nicole M Hoitsma ◽  
Amy M Whitaker ◽  
Emily C Beckwitt ◽  
Sunbok Jang ◽  
Pratul K Agarwal ◽  
...  
Keyword(s):  
Active Site ◽  
Excision Repair ◽  
Backbone Cleavage ◽  
Tyrosine Residue ◽  
Ap Endonuclease ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
Ap Endonuclease 1 ◽  
Base Excision ◽  
Dynamics Simulations

Abstract Base excision repair (BER) maintains genomic stability through the repair of DNA damage. Within BER, AP-endonuclease 1 (APE1) is a multifunctional enzyme that processes DNA intermediates through its backbone cleavage activity. To accomplish these repair activities, APE1 must recognize and accommodate several diverse DNA substrates. This is hypothesized to occur through a DNA sculpting mechanism where structural adjustments of the DNA substrate are imposed by the protein; however, how APE1 uniquely sculpts each substrate within a single rigid active site remains unclear. Here, we utilize structural and biochemical approaches to probe the DNA sculpting mechanism of APE1, specifically by characterizing a protein loop that intercalates the minor groove of the DNA (termed the intercalating loop). Pre-steady-state kinetics reveal a tyrosine residue within the intercalating loop (Y269) that is critical for AP-endonuclease activity. Using X-ray crystallography and molecular dynamics simulations, we determined the Y269 residue acts to anchor the intercalating loop on abasic DNA. Atomic force microscopy reveals the Y269 residue is required for proper DNA bending by APE1, providing evidence for the importance of this mechanism. We conclude that this previously unappreciated tyrosine residue is key to anchoring the intercalating loop and stabilizing the DNA in the APE1 active site.

scholarly journals A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase β

2010 ◽  
Vol 39 (8) ◽  
pp. 3156-3165 ◽  
Author(s):  
De-Sheng Pei ◽  
Xiao-Jie Yang ◽  
Wei Liu ◽  
Jeroen E. J. Guikema ◽  
Carol E. Schrader ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Polymerase Β ◽  
Ap Endonuclease ◽  
Regulatory Circuit ◽  
Ap Endonuclease 1 ◽  
Base Excision

scholarly journals AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair

2015 ◽  
Vol 43 (12) ◽  
pp. 5948-5960 ◽  
Author(s):  
Jill M. Beaver ◽  
Yanhao Lai ◽  
Meng Xu ◽  
Astrid H. Casin ◽  
Eduardo E. Laverde ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Trinucleotide Repeat ◽  
Excision Repair ◽  
Repeat Expansion ◽  
Trinucleotide Repeat Expansion ◽  
Ap Endonuclease ◽  
Ap Endonuclease 1 ◽  
Base Excision

scholarly journals Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

2021 ◽  
Vol 22 (9) ◽  
pp. 4769
Author(s):  
Pablo Maturana ◽  
María S. Orellana ◽  
Sixto M. Herrera ◽  
Ignacio Martínez ◽  
Maximiliano Figueroa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Conformational Dynamics ◽  
High Specificity ◽  
Arginine Decarboxylase ◽  
Space Groups ◽  
X Ray Crystallography ◽  
High Dynamics ◽  
Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

scholarly journals Molecular mechanisms associated with clustered lesion-induced impairment of 8-oxoG recognition by the human glycosylase OGG1

2021 ◽  
Author(s):  
Tao Jiang ◽  
Antonio MONARI ◽  
Elise Dumont ◽  
Emmanuelle Bignon
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Excision Repair ◽  
Structural Features ◽  
Repair Pathway ◽  
Dna Lesion ◽  
Damage Response ◽  
Base Excision ◽  
Structural Signature ◽  
Dynamics Simulations

The 8-oxo-7,8-dihydroguanine, referred to as 8-oxoG, is a highly mutagenic DNA lesion that can provoke the appearance of mismatches if it escapes the DNA Damage Response. The specific recognition of its structural signature by the hOGG1 glycosylase is the first step along the Base Excision Repair pathway, that ensures the integrity of the genome by preventing the emergence of mutations. 8-oxoG formation, structural features and repair have been the matter of extensive research and more recently this active field of research expended to the more complicated case of 8-oxoG within clustered lesions. Indeed, the presence of a second lesion within 1 or 2 helix turns can dramatically impact the repair yields of 8-oxoG by glycosylases. In this work, we use mu-range molecular dynamics simulations and machine learning-based post-analysis to explore the molecular mechanisms associated with the recognition of 8-oxoG by hOGG1 when embedded in a multiple lesions site with a mismatch in 5' or 3'. We delineate the stiffening of the DNA-protein interactions upon the presence of the mismatches, and rationalize the much lower repair yields reported with a 5' mismatch by describing the perturbation of 8-oxoG structural features upon addition of an adjacent lesion.

Studies on the base excision repair (BER) complex in Pyrococcus furiosus

2009 ◽  
Vol 37 (1) ◽  
pp. 79-82 ◽  
Author(s):  
Shinichi Kiyonari ◽  
Saki Tahara ◽  
Maiko Uchimura ◽  
Tsuyoshi Shirai ◽  
Sonoko Ishino ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Pyrococcus Furiosus ◽  
Excision Repair ◽  
Repair Process ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Ap Endonuclease ◽  
Reading Frame ◽  
Base Excision

We have been studying the functions of PCNA (proliferating-cell nuclear antigen) for the assembly and reassembly of the replisome during replication fork progression. We have identified the functional interactions between PCNA and several proteins involved in DNA replication and repair from Pyrococcus furiosus. We recently reported that the activity of UDG (uracil–DNA glycosylase) in P. furiosus (PfuUDG) is stimulated by PCNA (PfuPCNA) in vitro, and identified an atypical PCNA-binding site, AKTLF, in the PfuUDG protein. To understand further the function of the complex in the BER (base excision repair) process, we investigated the AP (apurinic/apyrimidinic) endonuclease, which can process the BER pathway after uracil removal by UDG. Interestingly, one candidate ORF (open reading frame) for the AP endonuclease was found in the operon containing the gene encoding UDG in the P. furiosus genome. However, this ORF did not exhibit any activity. Instead, we identified the AP endonuclease activity from the other candidate gene products, and designated the protein as PfuAP. We discovered a physical interaction between PfuAP and PfuPCNA, suggesting the formation of a BER complex in one of the repair systems in P. furiosus.

scholarly journals Spontaneous Mutagenesis Is Enhanced in Apex Heterozygous Mice

2004 ◽  
Vol 24 (18) ◽  
pp. 8145-8153 ◽  
Author(s):  
Jessica Huamani ◽  
C. Alex McMahan ◽  
Damon C. Herbert ◽  
Robert Reddick ◽  
John R. McCarrey ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Germ Line ◽  
Excision Repair ◽  
Spontaneous Mutant ◽  
Spermatogenic Cells ◽  
Ap Endonuclease ◽  
Repair Activity ◽  
Base Excision ◽  
Mutant Frequency

ABSTRACT Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex +/−) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex +/− lacI + mice compared to frequencies from Apex +/+ lacI + littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex +/− lacI + mice were significantly elevated twofold compared to levels for 9-month-old Apex +/+ lacI + control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.

scholarly journals Bending of DNA duplexes with mutation motifs

DNA Research ◽  
2019 ◽  
Vol 26 (4) ◽  
pp. 341-352
Author(s):  
Michal Růžička ◽  
Přemysl Souček ◽  
Petr Kulhánek ◽  
Lenka Radová ◽  
Lenka Fajkusová ◽  
...  
Keyword(s):  
De Novo ◽  
Excision Repair ◽  
Dna Duplexes ◽  
De Novo Mutations ◽  
Base Pairs ◽  
Inherited Disorders ◽  
Cpg Dinucleotides ◽  
Human Genes ◽  
Base Excision ◽  
Dynamics Simulations

Abstract Mutations can be induced by environmental factors but also arise spontaneously during DNA replication or due to deamination of methylated cytosines at CpG dinucleotides. Sites where mutations occur with higher frequency than would be expected by chance are termed hotspots while sites that contain mutations rarely are termed coldspots. Mutations are permanently scanned and repaired by repair systems. Among them, the mismatch repair targets base pair mismatches, which are discriminated from canonical base pairs by probing altered elasticity of DNA. Using biased molecular dynamics simulations, we investigated the elasticity of coldspots and hotspots motifs detected in human genes associated with inherited disorders, and also of motifs with Czech population hotspots and de novo mutations. Main attention was paid to mutations leading to G/T and A+/C pairs. We observed that hotspots without CpG/CpHpG sequences are less flexible than coldspots, which indicates that flexible sequences are more effectively repaired. In contrary, hotspots with CpG/CpHpG sequences exhibited increased flexibility as coldspots. Their mutability is more likely related to spontaneous deamination of methylated cytosines leading to C > T mutations, which are primarily targeted by base excision repair. We corroborated conclusions based on computer simulations by measuring melting curves of hotspots and coldspots containing G/T mismatch.

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