A Mathematical Model for Scanning and Catalysis on Single-stranded DNA, Illustrated with Activation-induced Deoxycytidine Deaminase

Mak, C. H.; Pham, Phuong; Afif, Samir A.; Goodman, Myron F.

doi:10.1074/jbc.m113.506550

Cited by 28 publications

(63 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Considering all states of the catalytic pocket regardless of dC accessibility, our modeling and docking results lead us to estimate that 1.3% of all AID-DNA interactions have the potential to result in cytidine deamination. These calculations derived solely from our in silico observations are in good agreement with a recent study that calculated deamination-conducive ssDNA binding events by AID in solution, derived from enzyme kinetic assays (Mak et al, 2013). This study suggested that 0.7%-8.0% of AID-DNA interactions result in dC deamination.…”

Section: The Role Of Dna Binding In Deamination Catalysissupporting

confidence: 90%

Catalytic Pocket Inaccessibility of Activation-Induced Cytidine Deaminase Is a Safeguard against Excessive Mutagenic Activity

et al. 2015

View full text Add to dashboard Cite

Activation-induced cytidine deaminase (AID) mutates cytidine to uridine at immunoglobulin loci to initiate secondary antibody diversification but also causes genome-wide damage. We previously demonstrated that AID has a relatively low catalytic rate. The structure of AID has not been solved. Thus, to probe the basis for its catalytic lethargy we generated a panel of free or DNA-bound AID models based on eight recently resolved APOBEC structures. Docking revealed that the majority of AID:DNA complexes would be inactive due to substrate binding such that a cytidine is not positioned for deamination. Furthermore, we found that most AID conformations exhibit fully or partially occluded catalytic pockets. We constructed mutant and chimeric AID variants predicted to have altered catalytic pocket accessibility dynamics and observed significant correlation with catalytic rate. Data from modeling simulations and functional tests of AID variants support the notion that catalytic pocket accessibility is an inherent bottleneck for AID activity.

show abstract

Section: The Role Of Dna Binding In Deamination Catalysissupporting

confidence: 90%

Catalytic Pocket Inaccessibility of Activation-Induced Cytidine Deaminase Is a Safeguard against Excessive Mutagenic Activity

et al. 2015

View full text Add to dashboard Cite

show abstract

“…It is unlikely that they are physical entry sites for the binding of AID because biochemical studies have shown that AID can bind equally well to single-stranded DNA that does and does not have Cs (67,68). However, AID is a very inefficient enzyme deaminating only one of every 30 dCs it encounters in hotspot motifs (57), so it is possible that the AGCTs in IGHV3-23*01 are often the first site to be deaminated. This is consistent with their being the most frequent site of mutation, but they might sometimes also act as an activation site to extend AID mutation to other sites in some V regions.…”

Section: Discussionmentioning

confidence: 99%

“…These studies confirmed that purified AID had a preference for the AGCT, even on single-stranded DNA. However, it is difficult to extrapolate the in vitro findings to the in vivo events because in the biochemical system, AID both is limiting and is processive through a slide and jump mechanism (56,57). The Ramos cells provide a good cellular model for the initial mutational events that occur in vivo because AID is constantly expressed and the relative susceptibility of the different AID hotspots to mutation is similar to that seen in mice (69).…”

Section: Discussionmentioning

confidence: 99%

“…Others have also seen differences between biochemical studies of the top (untranscribed) and bottom (transcribed) strands and attributed these to the fact that the in vivo bottom strand is heavily occupied by RNA polymerase and its many associated factors, including exosomes, so it is more difficult to duplicate the process biochemically (14,51). Because AID is limited and processive through a slide-and-jump mechanism in this biochemical assay (56,57), these results suggest that even on single-stranded DNA, the AGCT motifs were more likely to undergo AID-induced deamination and, through AID's processivity in this in vitro system, increase the frequency of mutation throughout the V region.…”

Section: Association Of Mutations In the Ohs1 And Ohs2 With Mutationsmentioning

confidence: 99%

See 1 more Smart Citation

Overlapping hotspots in CDRs are critical sites for V region diversification

Wei

Chahwan

Wang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Activation-induced deaminase (AID) mediates the somatic hypermutation (SHM) of Ig variable (V) regions that is required for the affinity maturation of the antibody response. An intensive analysis of a published database of somatic hypermutations that arose in the IGHV3-23*01 human V region expressed in vivo by human memory B cells revealed that the focus of mutations in complementary determining region (CDR)1 and CDR2 coincided with a combination of overlapping AGCT hotspots, the absence of AID cold spots, and an abundance of polymerase eta hotspots. If the overlapping hotspots in the CDR1 or CDR2 did not undergo mutation, the frequency of mutations throughout the V region was reduced. To model this result, we examined the mutation of the human IGHV3-23*01 biochemically and in the endogenous heavy chain locus of Ramos B cells. Deep sequencing revealed that IGHV3-23*01 in Ramos cells accumulates AID-induced mutations primarily in the AGCT in CDR2, which was also the most frequent site of mutation in vivo. Replacing the overlapping hotspots in CDR1 and CDR2 with neutral or cold motifs resulted in a reduction in mutations within the modified motifs and, to some degree, throughout the V region. In addition, some of the overlapping hotspots in the CDRs were at sites in which replacement mutations could change the structure of the CDR loops. Our analysis suggests that the local sequence environment of the V region, and especially of the CDR1 and CDR2, is highly evolved to recruit mutations to key residues in the CDRs of the IgV region.A fter an encounter with antigen and subsequent migration into the germinal centers of the secondary lymphoid organs, B cells undergo a regulated cascade of mutational events that occur at a very high frequency and are largely restricted to the variable (V) and switch (S) regions of the Ig heavy chain locus and the V region of the light chain locus. These mutagenic events are responsible for the somatic hypermutation (SHM) of the V regions and the class switch recombination of the constant (C) regions that are required for protective antibodies (1, 2). Both SHM and class switch recombination are initiated by activationinduced deaminase (AID) that preferentially deaminates the dC residues in WRC (W = A/T, R = A/G) hotspot motifs at frequencies 2-10-fold higher than SYC (S = G/C; Y = C/T) cold spots (3-7). During V region SHM, the resulting dU:G mismatch can then be replicated during S-phase to produce transition mutations, be processed by uracil-DNA glycosylase 2 and apurinic/ apyrimidinic endonucleases through the base excision repair pathway to produce both transitions and transversions (8-10), or be recognized by MutS homolog (MSH)2/MSH6 of the mismatch repair (MMR) complex that recruits the low-fidelity polymerase eta (Polη) to generate additional mutations at neighboring A:T residues (11).The specificity of AID targeting to the Ig gene has been under intense investigation. Studies have shown that AID deamination and mutagenesis targets single-stranded DNA substrates generated during ...

show abstract

“…A3A activity is also enhanced by cooperative dimerization [265]. Further, the related family member Activation Induced cytidine Deaminase (AID) that deaminates within specific regions of immunoglobulin genes to promote somatic hypermutation and class switching has been characterized as being a catalytically inefficient enzyme [188,266,267]. The low efficiency of catalysis is despite AID's high processivity and ability to remain bound to ssDNA for an average of 5 min [268].…”

Section: Modulation Of Catalytic Activity In the Aid/apobec Familymentioning

confidence: 99%

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

2018

View full text Add to dashboard Cite

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...

show abstract

A Mathematical Model for Scanning and Catalysis on Single-stranded DNA, Illustrated with Activation-induced Deoxycytidine Deaminase

Cited by 28 publications

References 44 publications

Catalytic Pocket Inaccessibility of Activation-Induced Cytidine Deaminase Is a Safeguard against Excessive Mutagenic Activity

Catalytic Pocket Inaccessibility of Activation-Induced Cytidine Deaminase Is a Safeguard against Excessive Mutagenic Activity

Overlapping hotspots in CDRs are critical sites for V region diversification

Biochemical Basis of APOBEC3 Deoxycytidine Deaminase Activity on Diverse DNA Substrates

Contact Info

Product

Resources

About