Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity

Kouno, Takahide; Silvas, Tania V.; Hilbert, Brendan J.; Shandilya, Shivender M.D.; Bohn, Markus-Frederik; Kelch, Brian A.; Royer, William E.; Somasundaran, Mohan; Yilmaz, Neşe Kurt; Matsuo, Hiroshi; Schiffer, Celia A.

doi:10.1038/ncomms15024

Cited by 144 publications

(288 citation statements)

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“…An A3H structural model was generated to help distinguish between these possibilities and inform mutagenesis experiments (Figure 2A). Based on recent A3A/B-ssDNA co-crystal structures (Kouno et al, 2017; Shi et al, 2017), loop 1, loop 7, and active site residues define a positively charged region that is likely essential for A3H to bind single-stranded DNA and potentially also RNA ( i.e ., in this model, the two activities will be inseparable by mutagenesis; patch 1 in Figure 2A). …”

Section: Resultsmentioning

confidence: 99%

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism

et al. 2018

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SUMMARY Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA editing enzymes function in innate immunity by restricting the replication of viruses and transposons. APOBEC3H also contributes to cancer mutagenesis. Here we address the fundamental nature of RNA in regulating human APOBEC3H activities. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment enables strong DNA deaminase activity. RNA binding-defective mutants demonstrate clear separation-of-function by becoming DNA hypermutators. Biochemical and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. Additionally, APOBEC3H separation-of-function mutants show that RNA binding is required for cytoplasmic localization, packaging into HIV-1 particles, and antiviral activity. Overall, these results support a model in which structured RNA negatively regulates the potentially harmful DNA deamination activity of APOBEC3H, while at the same time positively regulating its antiviral activity.

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Section: Resultsmentioning

confidence: 99%

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism

et al. 2018

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“…The apparent discrepancy between the results in the apo structures and the cocomplexes may be explained by a new hypothesis we propose for the mechanism of substrate binding by A3A, in light of a third structure of an A3–substrate complex reported recently, where human A3F‐CTD was bound to a nonsubstrate Poly_T ssDNA. This complex identified a second nonspecific polynucleotide binding site away from the active site [Fig.…”

Section: Introductionmentioning

confidence: 97%

“…These results suggested that the homodimer interface seen in apo A3A crystal structure may mediate cooperative protein–protein interactions that affect A3A activity. However, cocrystal structures of A3A bound to ssDNA revealed a monomer in the asymmetric unit, with a 1:1 stoichiometry with ssDNA.…”

Section: Introductionmentioning

confidence: 99%

APOBEC3s: DNA‐editing human cytidine deaminases

Silvas

Schiffer

2019

Protein Science

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Nucleic acid editing enzymes are essential components of the human immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins. Among these enzymes are cytidine deaminases of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) super family, each with unique target sequence specificity and subcellular localization. We focus on the DNA‐editing APOBEC3 enzymes that have recently attracted attention because of their involvement in cancer and potential in gene‐editing applications. We review and compare the crystal structures of APOBEC3 (A3) domains, binding interactions with DNA, substrate specificity, and activity. Recent crystal structures of A3A and A3G bound to ssDNA have provided insights into substrate binding and specificity determinants of these enzymes. Still many unknowns remain regarding potential cooperativity, nucleic acid interactions, and systematic quantification of substrate preference of many APOBEC3s, which are needed to better characterize the biological functions and consequences of misregulation of these gene editors.

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“…It remains to be determined whether increased fl A3B activity is due to different catalytic pocket accessibility in the fl A3B than A3B CTD, if the NTD of A3B is better able to guide the ssDNA substrate into the catalytic pocket, if increased processivity enables a more efficient search for potential deamination motifs, or a combination of all of these factors. Recently, it has been shown that A3A and A3B CTD bend the ssDNA substrate in the active site [344,373,374]. How this relates to processivity and if the enzymes can induce the ssDNA bending of larger substrates, such as dynamic ssDNA exposed during replication or transcription remains to be studied.…”

Section: Discussionmentioning

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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Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity

Cited by 144 publications

References 72 publications

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by an RNA-Mediated Dimerization Mechanism

APOBEC3s: DNA‐editing human cytidine deaminases

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

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