scholarly journals Functional Roles of PARP2 in Assembling Protein–Protein Complexes Involved in Base Excision DNA Repair

2021 ◽  
Vol 22 (9) ◽  
pp. 4679
Author(s):  
Inna Vasil’eva ◽  
Nina Moor ◽  
Rashid Anarbaev ◽  
Mikhail Kutuzov ◽  
Olga Lavrik
Keyword(s):  
Excision Repair ◽  
Protein Complexes ◽  
Binding Affinities ◽  
Reaction Conditions ◽  
Functional Roles ◽  
Affinity Constants ◽  
Protein Partners ◽  
Independent Mechanism ◽  
Base Excision ◽  
Base Excision Dna Repair

Poly(ADP-ribose) polymerase 2 (PARP2) participates in base excision repair (BER) alongside PARP1, but its functions are still under study. Here, we characterize binding affinities of PARP2 for other BER proteins (PARP1, APE1, Polβ, and XRCC1) and oligomerization states of the homo- and hetero-associated complexes using fluorescence-based and light scattering techniques. To compare PARP2 and PARP1 in the efficiency of PAR synthesis, in the absence and presence of protein partners, the size of PARP2 PARylated in various reaction conditions was measured. Unlike PARP1, PARP2 forms more dynamic complexes with common protein partners, and their stability is effectively modulated by DNA intermediates. Apparent binding affinity constants determined for homo- and hetero-oligomerized PARP1 and PARP2 provide evidence that the major form of PARP2 at excessive PARP1 level is their heterocomplex. Autoregulation of PAR elongation at high PARP and NAD+ concentrations is stronger for PARP2 than for PARP1, and the activity of PARP2 is more effectively inhibited by XRCC1. Moreover, the activity of both PARP1 and PARP2 is suppressed upon their heteroPARylation. Taken together, our findings suggest that PARP2 can function differently in BER, promoting XRCC1-dependent repair (similarly to PARP1) or an alternative XRCC1-independent mechanism via hetero-oligomerization with PARP1.

scholarly journals Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

2016 ◽  
Vol 113 (28) ◽  
pp. 7792-7797 ◽  
Author(s):  
Chenxu Zhu ◽  
Lining Lu ◽  
Jun Zhang ◽  
Zongwei Yue ◽  
Jinghui Song ◽  
...  
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Mammalian Genome ◽  
Dna Bases ◽  
Repair Pathway ◽  
Thymine Glycol ◽  
Base Excision ◽  
Oxidation Damage ◽  
Biochemical Analyses ◽  
Base Excision Dna Repair

NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)—a preferred substrate—for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzyme-promoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.

Scanning Force Microscopy and Nanomangulation: Studies of Dna and Proteins Involved in Dna Repair

1999 ◽  
Vol 5 (S2) ◽  
pp. 1004-1005
Author(s):  
Dorothy Erie ◽  
Glenn Ratcliff ◽  
Martin Guthold ◽  
Valerie Bullock ◽  
Michelle Pliske ◽  
...  
Keyword(s):  
Dna Repair ◽  
Physical Properties ◽  
Conformational Changes ◽  
Excision Repair ◽  
Protein Complexes ◽  
Scanning Force Microscopy ◽  
Force Microscopy ◽  
Scanning Force ◽  
Base Excision ◽  
User Friendly

Repair of damaged or incorrectly matched DNA is essential to the survival of all organisms. Consequently cells have devised a plentitude of pathways for repair. We have been investigating the mechanisms of mismatch repair and base excision repair. Both of these repair processes involve a large number of proteins that interact with one another as well as with DNA. Our long-term goal is to assemble complexes that are fully functional for DNA repair and to image the process of DNA repair. In addition, we wish to i) determine the stoicheometry of binding of the protein complexes to each other and to DNA, ii) monitor conformational changes due to substrate binding, iii) measure physical properties of DNA and the complexes. To accomplish this end, we have endeavored to improve techniques for solution imaging as well as those for data analysis. In this presentation I will discuss data on the stoicheometry of binding in several protein complexes and data on the physical properties of DNA.To measure the physical properties of DNA, we utilize a nanoManipulator, a modified Scanning Force Microscope with a novel, user-friendly interface that allows easy and controlled manipulation of nanometer-sized samples.

scholarly journals Quantitative characterization of protein–protein complexes involved in base excision DNA repair

2015 ◽  
Vol 43 (12) ◽  
pp. 6009-6022 ◽  
Author(s):  
Nina A. Moor ◽  
Inna A. Vasil'eva ◽  
Rashid O. Anarbaev ◽  
Alfred A. Antson ◽  
Olga I. Lavrik
Keyword(s):  
Dna Repair ◽  
Protein Complexes ◽  
Quantitative Characterization ◽  
Base Excision ◽  
Base Excision Dna Repair

Base excision DNA repair in the embryonic development of the sea urchin, Strongylocentrotus intermedius

2016 ◽  
Vol 12 (7) ◽  
pp. 2247-2256 ◽  
Author(s):  
Natalya A. Torgasheva ◽  
Natalya I. Menzorova ◽  
Yurii T. Sibirtsev ◽  
Valery A. Rasskazov ◽  
Dmitry O. Zharkov ◽  
...  
Keyword(s):  
Dna Repair ◽  
Embryonic Development ◽  
Sea Urchin ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Strongylocentrotus Intermedius ◽  
Stages Of Development ◽  
Base Excision ◽  
Base Excision Dna Repair

We have characterized the profile of several key base excision repair activities in the developing embryo of the grey sea urchin,Strongylocentrotus intermedius, at several stages of development.

scholarly journals How are base excision DNA repair pathways deployed in vivo?

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 279 ◽  
Author(s):  
Upasna Thapar ◽  
Bruce Demple
Keyword(s):  
Dna Repair ◽  
Base Excision Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Biochemical Level ◽  
Repair Pathways ◽  
Base Excision ◽  
Base Excision Dna Repair

Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them.

Excision repair cross complementing 6 (ERCC6) single nucleotide polymorphism (SNP) and outcome to gemcitabine (gem)/cisplatin (cis) or docetaxel (doc)/cis in stage IV non-small cell lung cancer (NSCLC) patients (pts)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7605-7605
Author(s):  
O. Etxaniz ◽  
M. Provencio ◽  
J. Terrasa ◽  
A. Carrato ◽  
P. Lianes ◽  
...  
Keyword(s):  
Chemotherapy Regimen ◽  
Excision Repair ◽  
Performance Status ◽  
Stage Iv ◽  
Gender Performance ◽  
Differential Sensitivity ◽  
Taqman Assay ◽  
Base Excision ◽  
Nsclc Patients ◽  
Base Excision Dna Repair

7605 Background: ERCC6 (alternate name CSB) is involved in both transcription coupled and base excision DNA repair, and the ERCC6 C-6530>G SNP is involved in gene regulation. Different levels of ERCC6 mRNA expression have been observed in cells according to ERCC6–6530 genotype. Methods: We investigated the ERCC6 C-6530>G SNP in 309 stage IV NSCLC pts treated with doc/cis (196 pts) and gem/cis (113 pts). DNA was extracted from peripheral lymphocytes and Taqman assay was used for SNP typing. Results: Distribution of ERCC6 genotypes was: CC 113 pts (36.6%); CG 157 pts (50.8%); GG 39 pts (12.6%). No differences in genotype were observed according to age, gender, performance status (PS), histology, chemotherapy regimen or second-line treatment. Overall time to progression (TTP) was 5.4 months (m) and median survival (MS) 9.9 m. No differences in TTP or MS were observed according to ERCC6 SNP types. However, when pts were broken down by chemotherapy regimen, TTP was 7 m for 31 CC pts treated with gem/cis and 5.4 m for 71 CC pts treated with doc/cis (P=0.04) ( Table ). MS was longer for CC pts treated with gem/cis (11 m) than for CC pts treated with doc/cis (8.9 m) (P=0.46). Differences were also observed in pts with PS 0 and in younger pts. Conclusions: ERCC6 C-6530>G SNP may confer differential sensitivity to gem or doc in combination with cis. We hypothesize that ERCC6 6350 CC is a surrogate of ERCC6 transcript, where lower ERCC6 expression levels may increase the activity of gem/cis in comparison to doc/cis. No significant financial relationships to disclose. [Table: see text]

scholarly journals Lysines in the lyase active site of DNA polymerase β destabilize nonspecific DNA binding, facilitating searching and DNA gap recognition

2020 ◽  
Vol 295 (34) ◽  
pp. 12181-12187 ◽  
Author(s):  
Michael J. Howard ◽  
Julie K. Horton ◽  
Ming-Lang Zhao ◽  
Samuel H. Wilson
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Active Site ◽  
Excision Repair ◽  
Fluorescent Microscopy ◽  
Phosphate Removal ◽  
Binding Affinities ◽  
Binding Behavior ◽  
Lysine Residues ◽  
Base Excision

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

scholarly journals Temporal dynamics of base excision / single-strand break repair protein complex assembly and disassembly are modulated by the PARP1/NAD+/SIRT6 axis

2021 ◽  
Author(s):  
Christopher A. Koczor ◽  
Kate M. Saville ◽  
Joel F. Andrews ◽  
Jennifer Clark ◽  
Qingming Fang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Temporal Dynamics ◽  
Excision Repair ◽  
Protein Complexes ◽  
Strand Break ◽  
Complex Assembly ◽  
Single Strand Break ◽  
Repair Protein ◽  
Base Excision

SUMMARYAssembly and disassembly of DNA repair protein complexes at sites of DNA damage is essential to maintain genomic integrity. We investigated factors coordinating assembly of the base excision repair (BER) proteins, DNA polymerase β (Polβ) and XRCC1, to DNA lesion sites, identifying a new role for Polβ in regulating XRCC1 disassembly from DNA repair complexes and conversely, demonstrating Polβ’s dependence on XRCC1 for complex assembly. RealPAR, a genetically-encoded probe for live cell imaging of poly(ADP-ribose) (PAR), reveals that Polβ and XRCC1 require PAR for repair complex assembly and PAR degradation for disassembly. We find that BER complex assembly is further modulated by attenuation / augmentation of NAD+ biosynthesis. Finally, SIRT6 does not regulate PARP1 activation but impairs XRCC1 recruitment, leading to diminished Polβ abundance at sites of DNA damage. These findings highlight coordinated yet independent roles for both PARP1 and SIRT6 and their regulation by NAD+ bioavailability to facilitate BER.

scholarly journals Interaction of Thymine DNA Glycosylase with Oxidised 5-Methyl-cytosines in Their Amino- and Imino-Forms

Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5728
Author(s):  
Senta Volkenandt ◽  
Frank Beierlein ◽  
Petra Imhof
Keyword(s):  
Excision Repair ◽  
Conformational Dynamics ◽  
Dna Glycosylase ◽  
Binding Affinities ◽  
Thymine Dna Glycosylase ◽  
Free Dna ◽  
Methyl Cytosine ◽  
Base Excision ◽  
Dynamics Simulations ◽  
Hydrolysis Of

Thymine DNA Glycosylase (TDG) is an enzyme of the base excision repair mechanism and removes damaged or mispaired bases from DNA via hydrolysis of the glycosidic bond. Specificity is of high importance for such a glycosylase, so as to avoid the damage of intact DNA. Among the substrates reported for TDG are mispaired uracil and thymine but also formyl-cytosine and carboxyl-cytosine. Methyl-cytosine and hydroxylmethyl-cytosine are, in contrast, not processed by the TDG enzyme. We have in this work employed molecular dynamics simulations to explore the conformational dynamics of DNA carrying a formyl-cytosine or carboxyl-cytosine and compared those to DNA with the non-cognate bases methyl-cytosine and hydroxylmethyl-cytosine, as amino and imino tautomers. Whereas for the mispairs a wobble conformation is likely decisive for recognition, all amino tautomers of formyl-cytosine and carboxyl-cytosine exhibit the same Watson–Crick conformation, but all imino tautomers indeed form wobble pairs. The conformational dynamics of the amino tautomers in free DNA do not exhibit differences that could be exploited for recognition, and also complexation to the TDG enzyme does not induce any alteration that would indicate preferable binding to one or the other oxidised methyl-cytosine. The imino tautomers, in contrast, undergo a shift in the equilibrium between a closed and a more open, partially flipped state, towards the more open form upon complexation to the TDG enzyme. This stabilisation of the more open conformation is most pronounced for the non-cognate bases methyl-cytosine and hydroxyl-cytosine and is thus not a likely mode for recognition. Moreover, calculated binding affinities for the different forms indicate the imino forms to be less likely in the complexed DNA. These findings, together with the low probability of imino tautomers in free DNA and the indifference of the complexed amino tautomers, suggest that discrimination of the oxidised methyl-cytosines does not take place in the initial complex formation.

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