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Article ; Online: Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A.

Kurup, Harikrishnan M / Kvach, Maksim V / Harjes, Stefan / Jameson, Geoffrey B / Harjes, Elena / Filichev, Vyacheslav V

2023 Volume 21, Issue 24, Page(s) 5117–5128

Abstract: The APOBEC3 (APOBEC3A-H) enzyme family as a part of the human innate immune system deaminates cytosine to uracil in single-stranded DNA (ssDNA) and thereby prevents the spread of pathogenic genetic information. However, APOBEC3-induced mutagenesis ... ...

Abstract	The APOBEC3 (APOBEC3A-H) enzyme family as a part of the human innate immune system deaminates cytosine to uracil in single-stranded DNA (ssDNA) and thereby prevents the spread of pathogenic genetic information. However, APOBEC3-induced mutagenesis promotes viral and cancer evolution, thus enabling the progression of diseases and development of drug resistance. Therefore, APOBEC3 inhibition offers a possibility to complement existing antiviral and anticancer therapies and prevent the emergence of drug resistance, thus making such therapies effective for longer periods of time. Here, we synthesised nucleosides containing seven-membered nucleobases based on azepinone and compared their inhibitory potential against human cytidine deaminase (hCDA) and APOBEC3A with previously described 2'-deoxyzebularine (dZ) and 5-fluoro-2'-deoxyzebularine (FdZ). The nanomolar inhibitor of wild-type APOBEC3A was obtained by the incorporation of 1,3,4,7-tetrahydro-2
MeSH term(s)	Humans ; Proteins/metabolism ; Cytidine Deaminase ; Mutagenesis ; Neoplasms/genetics ; Minor Histocompatibility Antigens
Chemical Substances	APOBEC3A protein, human (EC 3.5.4.5) ; Proteins ; Cytidine Deaminase (EC 3.5.4.5) ; APOBEC3B protein, human (EC 3.5.4.5) ; Minor Histocompatibility Antigens
Language	English
Publishing date	2023-06-21
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2097583-1
ISSN	1477-0539 ; 1477-0520
ISSN (online)	1477-0539
ISSN	1477-0520
DOI	10.1039/d3ob00392b
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Article: Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

Harjes, Stefan / Kurup, Harikrishnan M / Rieffer, Amanda E / Bayaijargal, Maitsetseg / Filitcheva, Jana / Su, Yongdong / Hale, Tracy K / Filichev, Vyacheslav V / Harjes, Elena / Harris, Reuben S / Jameson, Geoffrey B

bioRxiv : the preprint server for biology

2023

Abstract: The normally antiviral enzyme ... ...

Abstract	The normally antiviral enzyme APOBEC3A
Language	English
Publishing date	2023-02-17
Publishing country	United States
Document type	Preprint
DOI	10.1101/2023.02.17.528918
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Article ; Online: Design, Synthesis, and Evaluation of a Cross-Linked Oligonucleotide as the First Nanomolar Inhibitor of APOBEC3A.

Kurup, Harikrishnan M / Kvach, Maksim V / Harjes, Stefan / Barzak, Fareeda M / Jameson, Geoffrey B / Harjes, Elena / Filichev, Vyacheslav V

Biochemistry

2022 Volume 61, Issue 22, Page(s) 2568–2578

Abstract: Drug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance ...

Abstract	Drug resistance is a major problem associated with anticancer chemo- and immunotherapies. Recent advances in the understanding of resistance mechanisms have revealed that enzymes of the APOBEC3 (A3) family contribute to the development of drug resistance in multiple cancers. A3 enzymes are polynucleotide cytidine deaminases that convert cytosine to uracil (C→U) in single-stranded DNA (ssDNA) and in this way protect humans against viruses and mobile retroelements. On the other hand, cancer cells use A3s, especially A3A and A3B, to mutate human DNA, and thus by increasing rates of evolution, cancer cells escape adaptive immune responses and resist drugs. However, as A3A and A3B are non-essential for primary metabolism, their inhibition opens up a strategy to augment existing anticancer therapies and suppress cancer evolution. To test our hypothesis that pre-shaped ssDNA mimicking the
MeSH term(s)	Humans ; Oligonucleotides ; Cytidine Deaminase/metabolism ; DNA, Single-Stranded ; Cytidine/chemistry ; Cytosine
Chemical Substances	APOBEC3A protein, human (EC 3.5.4.5) ; Oligonucleotides ; Cytidine Deaminase (EC 3.5.4.5) ; DNA, Single-Stranded ; Cytidine (5CSZ8459RP) ; Cytosine (8J337D1HZY)
Language	English
Publishing date	2022-10-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1108-3
ISSN	1520-4995 ; 0006-2960
ISSN (online)	1520-4995
ISSN	0006-2960
DOI	10.1021/acs.biochem.2c00449
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Article ; Online: Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.

Harjes, Stefan / Kurup, Harikrishnan M / Rieffer, Amanda E / Bayarjargal, Maitsetseg / Filitcheva, Jana / Su, Yongdong / Hale, Tracy K / Filichev, Vyacheslav V / Harjes, Elena / Harris, Reuben S / Jameson, Geoffrey B

Nature communications

2023 Volume 14, Issue 1, Page(s) 6382

Abstract: The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA ... ...

Abstract	The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.
MeSH term(s)	Humans ; Proteins/chemistry ; Mutagenesis ; Neoplasms/drug therapy ; Neoplasms/genetics ; Neoplasms/pathology ; DNA ; Cytidine Deaminase/genetics ; Cytidine Deaminase/chemistry
Chemical Substances	APOBEC3A protein, human (EC 3.5.4.5) ; Proteins ; DNA (9007-49-2) ; Cytidine Deaminase (EC 3.5.4.5)
Language	English
Publishing date	2023-10-11
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-023-42174-w
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Article ; Online: Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity.

Barzak, Fareeda M / Ryan, Timothy M / Mohammadzadeh, Nazanin / Harjes, Stefan / Kvach, Maksim V / Kurup, Harikrishnan M / Krause, Kurt L / Chelico, Linda / Filichev, Vyacheslav V / Harjes, Elena / Jameson, Geoffrey B

Viruses

2022 Volume 14, Issue 9

Abstract: APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although ... ...

Abstract	APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.
MeSH term(s)	APOBEC-3G Deaminase/metabolism ; Cytidine Deaminase ; Cytosine ; DNA, Single-Stranded ; Deoxycytidine ; Polynucleotides ; Protein Binding ; Proteins ; RNA/metabolism ; Retroelements ; Scattering, Small Angle ; Uracil ; X-Ray Diffraction ; X-Rays
Chemical Substances	DNA, Single-Stranded ; Polynucleotides ; Proteins ; Retroelements ; Deoxycytidine (0W860991D6) ; Uracil (56HH86ZVCT) ; RNA (63231-63-0) ; Cytosine (8J337D1HZY) ; APOBEC-3G Deaminase (EC 3.5.4.5) ; APOBEC3A protein, human (EC 3.5.4.5) ; Cytidine Deaminase (EC 3.5.4.5)
Language	English
Publishing date	2022-09-06
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v14091974
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Article ; Online: Selective inhibition of APOBEC3 enzymes by single-stranded DNAs containing 2'-deoxyzebularine.

Barzak, Fareeda M / Harjes, Stefan / Kvach, Maksim V / Kurup, Harikrishnan M / Jameson, Geoffrey B / Filichev, Vyacheslav V / Harjes, Elena

Organic & biomolecular chemistry

2019 Volume 17, Issue 43, Page(s) 9435–9441

Abstract: To restrict pathogens, in a normal human cell, APOBEC3 enzymes mutate cytosine to uracil in foreign single-stranded DNAs. However, in cancer cells, APOBEC3B (one of seven APOBEC3 enzymes) has been identified as the primary source of genetic mutations. As ...

Abstract	To restrict pathogens, in a normal human cell, APOBEC3 enzymes mutate cytosine to uracil in foreign single-stranded DNAs. However, in cancer cells, APOBEC3B (one of seven APOBEC3 enzymes) has been identified as the primary source of genetic mutations. As such, APOBEC3B promotes evolution and progression of cancers and leads to development of drug resistance in multiple cancers. As APOBEC3B is a non-essential protein, its inhibition can be used to suppress emergence of drug resistance in existing anti-cancer therapies. Because of the vital role of APOBEC3 enzymes in innate immunity, selective inhibitors targeting only APOBEC3B are required. Here, we use the discriminative properties of wild-type APOBEC3A, APOBEC3B and APOBEC3G to deaminate different cytosines in the CCC-recognition motif in order to best place the cytidine analogue 2'-deoxyzebularine (dZ) in the CCC-motif. Using several APOBEC3 variants that mimic deamination patterns of wild-type enzymes, we demonstrate that selective inhibition of APOBEC3B in preference to other APOBEC3 constructs is feasible for the dZCC motif. This work is an important step towards development of in vivo tools to inhibit APOBEC3 enzymes in living cells by using short, chemically modified oligonucleotides.
MeSH term(s)	Cell Line ; Cytidine/analogs & derivatives ; Cytidine/chemistry ; Cytidine/pharmacology ; Cytidine Deaminase/antagonists & inhibitors ; Cytidine Deaminase/metabolism ; DNA, Single-Stranded/chemistry ; DNA, Single-Stranded/pharmacology ; Enzyme Inhibitors/chemical synthesis ; Enzyme Inhibitors/chemistry ; Enzyme Inhibitors/pharmacology ; Humans ; Molecular Structure ; Proteins/antagonists & inhibitors ; Proteins/metabolism
Chemical Substances	2'-deoxyzebularine ; DNA, Single-Stranded ; Enzyme Inhibitors ; Proteins ; Cytidine (5CSZ8459RP) ; APOBEC3A protein, human (EC 3.5.4.5) ; Cytidine Deaminase (EC 3.5.4.5)
Language	English
Publishing date	2019-10-08
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2097583-1
ISSN	1477-0539 ; 1477-0520
ISSN (online)	1477-0539
ISSN	1477-0520
DOI	10.1039/c9ob01781j
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Article ; Online: NMR-based method of small changes reveals how DNA mutator APOBEC3A interacts with its single-stranded DNA substrate.

Harjes, Stefan / Jameson, Geoffrey B / Filichev, Vyacheslav V / Edwards, Patrick J B / Harjes, Elena

Nucleic acids research

2017 Volume 45, Issue 9, Page(s) 5602–5613

Abstract: APOBEC3 proteins are double-edged swords. They deaminate cytosine to uracil in single-stranded DNA and provide protection, as part of our innate immune system, against viruses and retrotransposons, but they are also involved in cancer evolution and ... ...

Abstract	APOBEC3 proteins are double-edged swords. They deaminate cytosine to uracil in single-stranded DNA and provide protection, as part of our innate immune system, against viruses and retrotransposons, but they are also involved in cancer evolution and development of drug resistance. We report a solution-state model of APOBEC3A interaction with its single-stranded DNA substrate obtained with the 'method of small changes'. This method compares pairwise the 2D 15N-1H NMR spectra of APOBEC3A bearing a deactivating mutation E72A in the presence of 36 slightly different DNA substrates. From changes in chemical shifts of peptide N-H moieties, the positions of each nucleotide relative to the protein can be identified. This provided distance restraints for molecular-dynamic simulations to derive a 3-D molecular model of the APOBEC3A-ssDNA complex. The model reveals that loops 1 and 7 of APOBEC3A move to accommodate substrate binding, indicating an important role for protein-DNA dynamics. Overall, our method may prove useful to study other DNA-protein complexes where crystallographic techniques or full NMR structure calculations are hindered by weak binding or other problems. Subsequent to submission, an APOBEC3A structure with a bound DNA oligomer was published and coordinates released, which has provided an unbiased validation of the 'method of small changes'.
MeSH term(s)	Cytidine Deaminase/metabolism ; DNA, Single-Stranded/metabolism ; Fluorescence ; Humans ; Magnetic Resonance Spectroscopy ; Molecular Dynamics Simulation ; Mutation/genetics ; Oligonucleotides/metabolism ; Proteins/metabolism ; Substrate Specificity ; Thermodynamics
Chemical Substances	DNA, Single-Stranded ; Oligonucleotides ; Proteins ; APOBEC3A protein, human (EC 3.5.4.5) ; Cytidine Deaminase (EC 3.5.4.5)
Language	English
Publishing date	2017-05-26
Publishing country	England
Document type	Journal Article
ZDB-ID	186809-3
ISSN	1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
ISSN (online)	1362-4962 ; 1362-4954
ISSN	0301-5610 ; 0305-1048
DOI	10.1093/nar/gkx196
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Article ; Online: Experimentally based structural model of Yih1 provides insight into its function in controlling the key translational regulator Gcn2.

Harjes, Elena / Jameson, Geoffrey B / Tu, Yi-Hsuan / Burr, Natalie / Loo, Trevor S / Goroncy, Alexander K / Edwards, Patrick J B / Harjes, Stefan / Munro, Ben / Göbl, Christoph / Sattlegger, Evelyn / Norris, Gillian E

FEBS letters

2020 Volume 595, Issue 3, Page(s) 324–340

Abstract: Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in ... ...

Abstract	Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1-Gcn2 interaction by binding to Gcn1, thus preventing Gcn2-mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.
Language	English
Publishing date	2020-11-22
Publishing country	England
Document type	Journal Article
ZDB-ID	212746-5
ISSN	1873-3468 ; 0014-5793
ISSN (online)	1873-3468
ISSN	0014-5793
DOI	10.1002/1873-3468.13990
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Article: Experimentally based structural model of Yih1 provides insight into its function in controlling the key translational regulator Gcn2

Harjes, Elena / Jameson, Geoffrey B. / Tu, Yi‐Hsuan / Burr, Natalie / Loo, Trevor S. / Goroncy, Alexander K. / Edwards, Patrick J.B. / Harjes, Stefan / Munro, Ben / Göbl, Christoph / Sattlegger, Evelyn / Norris, Gillian E.

FEBS letters. 2021 Feb., v. 595, no. 3

2021

Abstract	Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1‐Gcn2 interaction by binding to Gcn1, thus preventing Gcn2‐mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.
Keywords	Saccharomyces cerevisiae ; cell cycle ; immune system ; models ; neurons ; nuclear magnetic resonance spectroscopy ; protein kinases ; protein synthesis ; yeasts
Language	English
Dates of publication	2021-02
Size	p. 324-340.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	JOURNAL ARTICLE
ZDB-ID	212746-5
ISSN	1873-3468 ; 0014-5793
ISSN (online)	1873-3468
ISSN	0014-5793
DOI	10.1002/1873-3468.13990
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Article: Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity

Barzak, Fareeda M. / Ryan, Timothy M. / Mohammadzadeh, Nazanin / Harjes, Stefan / Kvach, Maksim V. / Kurup, Harikrishnan M. / Krause, Kurt L. / Chelico, Linda / Filichev, Vyacheslav V. / Harjes, Elena / Jameson, Geoffrey B.

Viruses. 2022 Sept. 06, v. 14, no. 9

2022

Abstract	APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes.
Keywords	RNA ; X-radiation ; cytosine ; deamination ; enzymes ; gel chromatography ; innate immunity ; irradiation ; models ; retrotransposons ; single-stranded DNA ; small-angle X-ray scattering ; uracil
Language	English
Dates of publication	2022-0906
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v14091974
Database	NAL-Catalogue (AGRICOLA)

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