APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

Shlyakhtenko, Luda S.; Dutta, Samrat; Banga, Jaspreet; Li, Ming; Harris, Reuben S.; Lyubchenko, Yuri L.

doi:10.1038/srep15648

Cited by 20 publications

(32 citation statements)

References 37 publications

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“…In the AFM force spectroscopy approach, as described in ref 15 and schematically shown in Figure 1, ssDNA covalently attached to the surface is probed by A3A immobilized on the AFM probe via a flexible tether (see Materials and Methods for specifics). The tether (PEG linker) provides A3A with orientational freedom for interacting with ssDNA.…”

Section: Resultsmentioning

confidence: 99%

“…15 This suggests that unlike the two-Zn-coordinating domain enzyme, A3G, the one-domain A3A has a single binding mode with ssDNA. Moreover, the similar L c values for A3A and A3A-cys indicate that this mode is independent of the type of attachment of A3A to the probe because both the C- and N-terminal attachments have identical L c positions.…”

Section: Discussionmentioning

confidence: 99%

“…15 Briefly, the force–distance ( F – D ) curves were fitted with the Worm-Like Chain (WLC) model using the following formula:

F false(x false) = \frac{k_{B} T}{L_{normalp}} [\frac{1}{4} {(1 - \frac{x}{L_{c}})}^{- 2} - \frac{1}{4} + \frac{x}{L_{normalc}}]

where F ( x ) is the force at distance x , k B is the Boltzmann constant, T is the absolute temperature, and L p and L c are the persistence length and the contour length, respectively. The persistence length was allowed to vary because of the existence of two flexible linkers: the PEG linker from the functionalized AFM probe and the ssDNA from the surface.…”

Section: Methodsmentioning

confidence: 99%

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Single-Molecule Force Spectroscopy Studies of APOBEC3A–Single-Stranded DNA Complexes

Shlyakhtenko

Dutta

et al. 2016

Biochemistry

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APOBEC3A (A3A) inhibits the replication of a range of viruses and transposons and might also play a role in carcinogenesis. It is a single-domain deaminase enzyme that interacts with single-stranded DNA (ssDNA) and converts cytidines to uridines within specific trinucleotide contexts. Although there is abundant information that describes the potential biological activities of A3A, the interplay between binding ssDNA and sequence-specific deaminase activity remains controversial. Using a single-molecule atomic force microscopy spectroscopy approach developed by Shlyakhtenko et al. [(2015) Sci. Rep. 5, 15648], we determine the stability of A3A in complex with different ssDNA sequences. We found that the strength of the complex is sequence-dependent, with more stable complexes formed with deaminase-specific sequences. A correlation between the deaminase activity of A3A and the complex strength was identified. The ssDNA binding properties of A3A and those for A3G are also compared and discussed.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…15 Briefly, the force–distance ( F – D ) curves were fitted with the Worm-Like Chain (WLC) model using the following formula:

F false(x false) = \frac{k_{B} T}{L_{normalp}} [\frac{1}{4} {(1 - \frac{x}{L_{c}})}^{- 2} - \frac{1}{4} + \frac{x}{L_{normalc}}]

Section: Methodsmentioning

confidence: 99%

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Single-Molecule Force Spectroscopy Studies of APOBEC3A–Single-Stranded DNA Complexes

Shlyakhtenko

Dutta

et al. 2016

Biochemistry

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“…All measurements were done in HEPES buffer (10 mM HEPES, 100 mM NaCl, PH 7.0). The data analysis was performed as described 20, 35, 37, 38 . Briefly, DFS data was obtained for pulling experiments performed at different velocities (100-3000 nm/sec).…”

Section: Methodsmentioning

confidence: 99%

Probing of miniPEGγ-PNA–DNA Hybrid Duplex Stability with AFM Force Spectroscopy

Dutta

Armitage

Lyubchenko³

2016

Biochemistry

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Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplex. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. (2011) Journal of Organic Chemistry 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γMPPNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that γMPPNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ∼ 0.030 ± 0.01 sec-1 for γMPPNA-DNA hybrid duplex vs. 0.375 ± 0.18 sec-1 for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γMPPNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γMPPNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.

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“…To test this line of reasoning we used another processive and double deaminase domain A3 that has been extensively studied with regards to processivity and oligomerization [98,99,355,356]. A3G forms monomers and dimers in solution and further oligomerizes on ssDNA by binding cooperatively (Figure 10.7G, Table 10.1, and Figure 10.3E) [72,99,102].…”

Section: Processivity Is Not Required For Deamination During Transcrimentioning

confidence: 99%

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

2018

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Apolipoprotein B mRNA-editing enzyme-catalytic, polypeptide-like 3 (APOBEC3) enzymes are a family of single-stranded (ss)DNA cytosine deaminases that serve as a host restriction factors for retrotransposons and viruses that contain a ssDNA intermediate. While the APOBEC3 family was originally expanded to restrict endogenous retroelements, the majority of these targets are now inactivated and the family has been found to act on different targets, such as viral ssDNA as a mechanism of viral defense. Some of the family members also catalyze "offtarget" deaminations in human ssDNA and have been implicated in somatic mutagenesis that can lead to cancer.My PhD thesis research investigated the biochemical mechanisms underlying both the benefits and the risks of the A3 family of enzymes. The central hypothesis of this work is that A3 enzymes have evolved distinct biochemical mechanisms to deaminate ssDNA that differentiate A3 restriction of retroelements and retroviruses from A3-induced somatic mutagenesis.Therefore, we investigated the biochemical mechanisms the A3 enzymes utilize during restriction of Human Immunodeficiency Virus (HIV) in both deamination-dependent and deaminationindependent modes, as well as the unique mechanisms employed during mutation of genomic DNA. There are seven APOBEC3 members (A-H, excluding E) and of these, four members (A3D, A3F, A3G, A3H) have been identified to restrict HIV replication in CD4 T+ cells, and currently three members (A3A, A3B and A3H haplotype I) have been implicated in somatic mutagenesis.The APOBEC3 enzymes that restrict HIV replication function optimally in the absence of the HIV viral infectivity factor (Vif). Vif targets APOBEC3 for degradation via the proteasome in HIV infected cells. For APOBEC3s that are able to fortuitously escape Vif mediated degradation and become encapsidated, the enzymes can deaminate cytosines to form uracils in viral (-)DNA. Replication of (-)DNA to (+)DNA causes HIV-1 reverse transcriptase to incorporate adenines opposite uracils which creates C/G→T/A transition mutations. While restriction of HIV most commonly occurs through this deamination-dependent mechanism, APOBEC3s have also been identified to interfere with HIV reverse transcriptase processes in a deamination-independent manner, although this mechanism is secondary to the deaminationdependent mode. In order to restrict retroelements and viruses such as HIV, the A3 enzymes iii require an efficient processive ssDNA scanning mechanism that allows them to search for, and locate cytosines on ssDNA in the finite amount of time the substrate is available during formation of the double-stranded DNA provirus. To understand if this requirement was lost in A3 enzymes unable to restrict HIV, we studied A3C. Human A3C is not able to restrict HIV, in comparison to the more active gorilla and chimpanzee A3C orthologs that can restrict HIV, however an explanation for this difference was lacking. A polymorphism, S188I, in human A3C, which is found in less than 3% of the global population lead...

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APOBEC3G Interacts with ssDNA by Two Modes: AFM Studies

Cited by 20 publications

References 37 publications

Single-Molecule Force Spectroscopy Studies of APOBEC3A–Single-Stranded DNA Complexes

Single-Molecule Force Spectroscopy Studies of APOBEC3A–Single-Stranded DNA Complexes

Probing of miniPEGγ-PNA–DNA Hybrid Duplex Stability with AFM Force Spectroscopy

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

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