Nucleosomal embedding reshapes the dynamics of abasic sites

ABSTRACTApurinic/apyrimidinic (AP) sites are the most common DNA lesions, which benefit from a most efficient repair by the base excision pathway. The impact of losing a nucleobase on the conformation and dynamics of B-DNA is well characterized. Yet AP sites seem to present an entirely different chemistry in nucleosomal DNA, with lifetimes reduced up to 100-fold, and the much increased formation of covalent DNA-protein cross-links, refractory to repair. We report microsecond range, all-atom molecular dynamics simulations that capture the conformational dynamics of AP sites and their tetrahydrofuran analogs at two symmetrical positions within a nucleosome core particle, starting from a recent crystal structure. Different behaviours between the deoxyribo-based and tetrahydrofuran-type abasic sites are evidenced. The two solvent-exposed lesion sites present contrasted extrahelicities, revealing the crucial role of the position of a defect around the histone core. Our all-atom simulations also identify and quantify the occurrence of several spontaneous, non-covalent interactions between AP and positively-charged residues from the histones H2A and H2B tails that prefigure DNA-protein cross-links. This study paves the way towards an in silico mapping of DNA-protein cross-links.

Download Full-text

Nucleosomal embedding reshapes the dynamics of abasic sites

Scientific Reports ◽

10.1038/s41598-020-73997-y ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Emmanuelle Bignon ◽

Victor E. P. Claerbout ◽

Tao Jiang ◽

Christophe Morell ◽

Natacha Gillet ◽

...

Keyword(s):

Conformational Dynamics ◽

Experimental Studies ◽

Dna Lesions ◽

Nucleosome Core ◽

Abasic Sites ◽

Cross Links ◽

Ap Sites ◽

Base Excision ◽

Dynamics Simulations ◽

The Impact

Abstract Apurinic/apyrimidinic (AP) sites are the most common DNA lesions, which benefit from a most efficient repair by the base excision pathway. The impact of losing a nucleobase on the conformation and dynamics of B-DNA is well characterized. Yet AP sites seem to present an entirely different chemistry in nucleosomal DNA, with lifetimes reduced up to 100-fold, and the much increased formation of covalent DNA-protein cross-links leading to strand breaks, refractory to repair. We report microsecond range, all-atom molecular dynamics simulations that capture the conformational dynamics of AP sites and their tetrahydrofuran analogs at two symmetrical positions within a nucleosome core particle, starting from a recent crystal structure. Different behaviours between the deoxyribo-based and tetrahydrofuran-type abasic sites are evidenced. The two solvent-exposed lesion sites present contrasted extrahelicities, revealing the crucial role of the position of a defect around the histone core. Our all-atom simulations also identify and quantify the frequency of several spontaneous, non-covalent interactions between AP and positively-charged residues from the histones H2A and H2B tails that prefigure DNA-protein cross-links. Such an in silico mapping of DNA-protein cross-links gives important insights for further experimental studies involving mutagenesis and truncation of histone tails to unravel mechanisms of DPCs formation.

Download Full-text

A dynamic view of histone tails interaction with clustered abasic sites in a nucleosome core particle

10.1101/2021.02.16.431417 ◽

2021 ◽

Author(s):

Emmanuelle Bignon ◽

Natacha Gillet ◽

Tao Jiang ◽

Christophe Morell ◽

Elise Dumont

Keyword(s):

Dynamical Behavior ◽

Dna Lesions ◽

Histone Tails ◽

Systematic Assessment ◽

Nucleosome Core ◽

Abasic Sites ◽

Mutational Hotspots ◽

The One ◽

Dynamics Simulations ◽

Experimental Findings

AbstractApurinic/apyrimidinic sites are the most common DNA damage under physiological conditions. Yet, their structural and dynamical behavior within nucleosome core particles has just begun to be investigated, and show dramatic differences with the one of abasic sites in B-DNA. Clusters of two or more abasic sites are repaired even less efficiently and hence constitute hotspots of high mutagenicity notably due to enhanced double-strand breaks formation. Based on a X-ray structure of a 146-bp DNA wrapped onto a histone core, we investigate the structural behavior of two bistranded abasic sites positioned at mutational hotspots along microsecond-range molecular dynamics simulations. Our simulations allow us to probe histone tails interactions at clustered abasic sites locations, with a definitive assignment of the key residues in-volved in the NCP-catalyzed formation of DNA–protein cross-linking in line with recent experimental findings, and pave the way towards a systematic assessment of histone tails response to DNA lesions.

Download Full-text

How (5′S) and (5′R) 5′,8-Cyclo-2′-Deoxypurines Affect Base Excision Repair of Clustered DNA Damage in Nuclear Extracts of xrs5 Cells? A Biochemical Study

Cells ◽

10.3390/cells10040725 ◽

2021 ◽

Vol 10 (4) ◽

pp. 725

Author(s):

Karolina Boguszewska ◽

Michał Szewczuk ◽

Julia Kaźmierczak-Barańska ◽

Bolesław T. Karwowski

Keyword(s):

Base Excision Repair ◽

Excision Repair ◽

Genetic Material ◽

Dna Lesions ◽

The Other ◽

Base Pairs ◽

Ap Sites ◽

Base Excision ◽

The Impact ◽

Ap Site

The clustered DNA lesions (CDLs) are a characteristic feature of ionizing radiation’s impact on the human genetic material. CDLs impair the efficiency of cellular repair machinery, especially base excision repair (BER). When CDLs contain a lesion repaired by BER (e.g., apurinic/apyrimidinic (AP) sites) and a bulkier 5′,8-cyclo-2′-deoxypurine (cdPu), which is not a substrate for BER, the repair efficiency of the first one may be affected. The cdPus’ influence on the efficiency of nuclear BER in xrs5 cells have been investigated using synthetic oligonucleotides with bi-stranded CDL (containing (5′S) 5′,8-cyclo-2′-deoxyadenosine (ScdA), (5′R) 5′,8-cyclo-2′-deoxyadenosine (RcdA), (5′S) 5′,8-cyclo-2′-deoxyguanosine (ScdG) or (5′R) 5′,8-cyclo-2′-deoxyguanosine (RcdG) in one strand and an AP site in the other strand at different interlesion distances). Here, for the first time, the impact of ScdG and RcdG was experimentally tested in the context of nuclear BER. This study shows that the presence of RcdA inhibits BER more than ScdA; however, ScdG decreases repair level more than RcdG. Moreover, AP sites located ≤10 base pairs to the cdPu on its 5′-end side were repaired less efficiently than AP sites located ≤10 base pairs on the 3′-end side of cdPu. The strand with an AP site placed opposite cdPu or one base in the 5′-end direction was not reconstituted for cdA nor cdG. CdPus affect the repair of the other lesion within the CDL. It may translate to a prolonged lifetime of unrepaired lesions leading to mutations and impaired cellular processes. Therefore, future research should focus on exploring this subject in more detail.

Download Full-text

The DNA repair enzyme MUTYH potentiates cytotoxicity of the alkylating agent MNNG by interacting with abasic sites

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010497 ◽

2020 ◽

Vol 295 (11) ◽

pp. 3692-3707

Author(s):

Alan G. Raetz ◽

Douglas M. Banda ◽

Xiaoyan Ma ◽

Gege Xu ◽

Anisha N. Rajavel ◽

...

Keyword(s):

Dna Repair ◽

Alkylating Agent ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Dna Lesions ◽

Repair Enzyme ◽

Abasic Sites ◽

Human Lymphoblastoid Cell ◽

Ap Sites ◽

Base Excision

Higher expression of the human DNA repair enzyme MUTYH has previously been shown to be strongly associated with reduced survival in a panel of 24 human lymphoblastoid cell lines exposed to the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The molecular mechanism of MUTYH-enhanced MNNG cytotoxicity is unclear, because MUTYH has a well-established role in the repair of oxidative DNA lesions. Here, we show in mouse embryonic fibroblasts (MEFs) that this MNNG-dependent phenotype does not involve oxidative DNA damage and occurs independently of both O6-methyl guanine adduct cytotoxicity and MUTYH-dependent glycosylase activity. We found that blocking of abasic (AP) sites abolishes higher survival of Mutyh-deficient (Mutyh−/−) MEFs, but this blockade had no additive cytotoxicity in WT MEFs, suggesting the cytotoxicity is due to MUTYH interactions with MNNG-induced AP sites. We found that recombinant mouse MUTYH tightly binds AP sites opposite all four canonical undamaged bases and stimulated apurinic/apyrimidinic endonuclease 1 (APE1)-mediated DNA incision. Consistent with these observations, we found that stable expression of WT, but not catalytically-inactive MUTYH, enhances MNNG cytotoxicity in Mutyh−/− MEFs and that MUTYH expression enhances MNNG-induced genomic strand breaks. Taken together, these results suggest that MUTYH enhances the rapid accumulation of AP-site intermediates by interacting with APE1, implicating MUTYH as a factor that modulates the delicate process of base-excision repair independently of its glycosylase activity.

Download Full-text

Abasic Sites in the Transcribed Strand of Yeast DNA Are Removed by Transcription-Coupled Nucleotide Excision Repair

Molecular and Cellular Biology ◽

10.1128/mcb.00308-10 ◽

2010 ◽

Vol 30 (13) ◽

pp. 3206-3215 ◽

Cited By ~ 45

Author(s):

Nayun Kim ◽

Sue Jinks-Robertson

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Genome Integrity ◽

Genetic Studies ◽

Abasic Sites ◽

Dna And Rna ◽

Nucleotide Excision ◽

Ap Sites ◽

Base Excision ◽

Ap Site

ABSTRACT Abasic (AP) sites are potent blocks to DNA and RNA polymerases, and their repair is essential for maintaining genome integrity. Although AP sites are efficiently dealt with through the base excision repair (BER) pathway, genetic studies suggest that repair also can occur via nucleotide excision repair (NER). The involvement of NER in AP-site removal has been puzzling, however, as this pathway is thought to target only bulky lesions. Here, we examine the repair of AP sites generated when uracil is removed from a highly transcribed gene in yeast. Because uracil is incorporated instead of thymine under these conditions, the position of the resulting AP site is known. Results demonstrate that only AP sites on the transcribed strand are efficient substrates for NER, suggesting the recruitment of the NER machinery by an AP-blocked RNA polymerase. Such transcription-coupled NER of AP sites may explain previously suggested links between the BER pathway and transcription.

Download Full-text

Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes

Journal of Biological Chemistry ◽

10.1074/jbc.m115.665547 ◽

2015 ◽

Vol 290 (34) ◽

pp. 21067-21075 ◽

Cited By ~ 17

Author(s):

John M. Hinz ◽

Peng Mao ◽

Daniel R. McNeill ◽

David M. Wilson

Keyword(s):

Excision Repair ◽

Nuclease Activity ◽

Nucleosome Core Particle ◽

Nucleosome Core ◽

Efficient Processing ◽

Ap Sites ◽

Mammalian Enzyme ◽

Base Excision ◽

Dna Complex ◽

Ap Site

Non-coding apurinic/apyrimidinic (AP) sites are generated at high frequency in genomic DNA via spontaneous hydrolytic, damage-induced or enzyme-mediated base release. AP endonuclease 1 (APE1) is the predominant mammalian enzyme responsible for initiating removal of mutagenic and cytotoxic abasic lesions as part of the base excision repair (BER) pathway. We have examined here the ability of wild-type (WT) and a collection of variant/mutant APE1 proteins to cleave at an AP site within a nucleosome core particle. Our studies indicate that, in comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not simply due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced AP-DNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Notably, this incision defect of R237C and G241R was observed on a pre-assembled DNA glycosylase·AP-DNA complex as well. Our results suggest that the BER enzyme, APE1, has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms.

Download Full-text

Evidence for the Involvement of Nucleotide Excision Repair in the Removal of Abasic Sites in Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.20.10.3522-3528.2000 ◽

2000 ◽

Vol 20 (10) ◽

pp. 3522-3528 ◽

Cited By ~ 56

Author(s):

Carlos A. Torres-Ramos ◽

Robert E. Johnson ◽

Louise Prakash ◽

Satya Prakash

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Repair Process ◽

Dna Glycosylases ◽

Abasic Sites ◽

Skin Cancers ◽

Nucleotide Excision ◽

Progressive Neurodegeneration ◽

Ap Sites ◽

Base Excision

ABSTRACT In eukaryotes, DNA damage induced by ultraviolet light and other agents which distort the helix is removed by nucleotide excision repair (NER) in a fragment ∼25 to 30 nucleotides long. In humans, a deficiency in NER causes xeroderma pigmentosum (XP), characterized by extreme sensitivity to sunlight and a high incidence of skin cancers. Abasic (AP) sites are formed in DNA as a result of spontaneous base loss and from the action of DNA glycosylases involved in base excision repair. In Saccharomyces cerevisiae, AP sites are removed via the action of two class II AP endonucleases, Apn1 and Apn2. Here, we provide evidence for the involvement of NER in the removal of AP sites and show that NER competes with Apn1 and Apn2 in this repair process. Inactivation of NER in the apn1Δ orapn1Δ apn2Δ strain enhances sensitivity to the monofunctional alkylating agent methyl methanesulfonate and leads to further impairment in the cellular ability to remove AP sites. A deficiency in the repair of AP sites may contribute to the internal cancers and progressive neurodegeneration that occur in XP patients.

Download Full-text

Origin of Conformational Dynamics in a Globular Protein

10.1101/724286 ◽

2019 ◽

Author(s):

Adam M. Damry ◽

Marc M. Mayer ◽

Aron Broom ◽

Natalie K. Goto ◽

Roberto A. Chica

Keyword(s):

Conformational Dynamics ◽

Protein Structures ◽

Vital Role ◽

Globular Protein ◽

Conformational Exchange ◽

Solution Nmr ◽

Wild Type ◽

In The Wild ◽

Dynamics Simulations ◽

The Impact

AbstractProtein structures are dynamic, undergoing specific motions that can play a vital role in function. However, the link between primary sequence and conformational dynamics remains poorly understood. Here, we studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual substitutions of core residues in DANCER-3, a streptococcal protein G domain β1 (Gβ1) variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Using a combination of solution NMR experiments and molecular dynamics simulations, we demonstrate that only two mutations are necessary to create this conformational exchange, and that these mutations work synergistically, with one destabilizing the native Gβ1 structure and the other allowing two new conformational states to be accessed on the energy landscape. Overall, our results show how conformational dynamics can appear in a stable globular fold, a critical step in the molecular evolution of new dynamics-linked functions.

Download Full-text

Origin of conformational dynamics in a globular protein

Communications Biology ◽

10.1038/s42003-019-0681-2 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 2

Author(s):

Adam M. Damry ◽

Marc M. Mayer ◽

Aron Broom ◽

Natalie K. Goto ◽

Roberto A. Chica

Keyword(s):

Conformational Dynamics ◽

Protein Structures ◽

Vital Role ◽

Globular Protein ◽

Conformational Exchange ◽

Solution Nmr ◽

Native Structure ◽

In The Wild ◽

Dynamics Simulations ◽

The Impact

AbstractProtein structures are dynamic, undergoing motions that can play a vital role in function. However, the link between primary sequence and conformational dynamics remains poorly understood. Here, we studied how conformational dynamics can arise in a globular protein by evaluating the impact of individual core-residue substitutions in DANCER-3, a streptococcal protein G domain β1 variant that we previously designed to undergo a specific mode of conformational exchange that has never been observed in the wild-type protein. Using a combination of solution NMR experiments and molecular dynamics simulations, we demonstrate that only two mutations are necessary to create this conformational exchange, and that these mutations work synergistically, with one destabilizing the native structure and the other allowing two new conformational states to be accessed on the energy landscape. Overall, our results show how dynamics can appear in a stable globular fold, a critical step in the molecular evolution of dynamics-linked functions.

Download Full-text