APOBEC3G (A3G) restricts HIV-1 infection by catalyzingprocessive C 3 U deaminations on single-stranded DNA (ssDNA) with marked 3 3 5 deamination polarity. Here we show that A3G exists in oligomeric states whose composition is dictated primarily by interactions with DNA, with salt playing an important, yet secondary, role. Directional deaminations correlate with the presence of dimers, tetramers, and larger oligomers observed by atomic force microscopy, and random deaminations appear to correlate mainly with monomers. The presence of a 30-nt weakly deaminated "dead" zone located at the 3-ssDNA end implies the presence of a preferred asymmetric direction for A3G catalysis. Single turnover reaction rates reveal a salt-dependent inhibition of C deamination toward the 3-ssDNA region, offering a molecular basis underlying A3G deamination polarity. Presteady state analysis demonstrates rapid diffusionlimited A3G-ssDNA binding, a slower salt-dependent conformational change, possibly indicative of DNA wrapping, and long (5-15 min) protein-DNA complex lifetimes. We suggest that diverse A3G oligomerization modes contribute to the human immunodeficiency virus, type 1, proviral DNA mutational bias.In 2002, Sheehy et al.(1) determined that APOBEC3G (A3G), originally called CEM15, a proposed cytidine deaminase based on sequence analysis, is the nonpermissive host factor that blocks virion infectivity factor-defective (⌬vif) HIV-1 infection of T cells. The experimental determination that CEM15 was a cytidine deaminase belonging to the APOBEC family followed soon after with experiments demonstrating G 3 A-induced hypermutation of proviral DNA in a ⌬vif HIV-1 virion (2, 3). A3G has a duplicated deaminase domain structure, but only the C-terminal domain is responsible for the single-stranded DNA (ssDNA) 2 deamination activity (4, 5).Apart from A3G-catalyzed deamination, A3G may also have the capacity for blocking reverse transcription, (ϩ)-DNA synthesis, and provirus formation either by interacting with RNA or DNA of HIV-1 (6 -8) or by possibly interacting with HIV-1 proteins (9, 10). However, these noncatalytic effects on HIV inhibition may be attributable to the overexpression of A3G and may not be occurring during normal infection (11-13).It is important to bear in mind that any actions of A3G on DNA, catalytic and possibly noncatalytic, are balanced in vivo against cellular RNA binding, which forms a high molecular mass A3G-RNA complex, which may prevent A3G incorporation into virions (7, 14 -16), and against HIV RNA binding, which forms an intravirion A3G complex in which A3G must be activated by HIV RNase H for DNA deamination to ensue (16).We have shown that A3G-catalyzed deamination occurs processively while exhibiting a 3Ј 3 5Ј polarity favoring deamination toward the 5Ј-region of ssDNA (15). Directional deamination is an intrinsic property of A3G, occurring in the absence of an obvious source of energy (e.g. ATP or GTP) (15) and can in principle contribute to the HIV-1 G 3 A mutational bias, increasing in a 3Ј-direction ...

MutL alpha, the heterodimeric eukaryotic MutL homolog, is required for DNA mismatch repair (MMR) in vivo. It has been suggested that conformational changes, modulated by adenine nucleotides, mediate the interactions of MutL alpha with other proteins in the MMR pathway, coordinating the recognition of DNA mismatches by MutS alpha and the activation of MutL alpha with the downstream events that lead to repair. Thus far, the only evidence for these conformational changes has come from X-ray crystallography of isolated domains, indirect biochemical analyses, and comparison to other members of the GHL ATPase family to which MutL alpha belongs. Using atomic force microscopy (AFM), coupled with biochemical techniques, we demonstrate that adenine nucleotides induce large asymmetric conformational changes in full-length yeast and human MutL alpha and that these changes are associated with significant increases in secondary structure. These data reveal an ATPase cycle in which sequential nucleotide binding, hydrolysis, and release modulate the conformational states of MutL alpha.

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DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL's endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS-MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.DNA mismatch repair | MutS | MutL | FRET

Prostate-associated gene 4 (PAGE4) is a cancer/testis antigen that is typically restricted to the testicular germ cells but is aberrantly expressed in cancer. Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the developing prostate and is also a stress-response protein that is upregulated in response to cellular stress. PAGE4 interacts with c-Jun, which is activated by the stress-response kinase JNK1, and plays an important role in the development and pathology of the prostate gland. Here, we have identified homeodomain-interacting protein kinase 1 (HIPK1), also a component of the stress-response pathway, as a kinase that phosphorylates PAGE4 at T51. We show that phosphorylation of PAGE4 is critical for its transcriptional activity since mutating this T residue abolishes its ability to potentiate c-Jun transactivation. In vitro single molecule FRET indicates phosphorylation results in compaction of (still) intrinsically disordered PAGE4. Interestingly, however, while our previous observations indicated that the wild-type nonphosphorylated PAGE4 protein interacted with c-Jun [RajagopalanK.RajagopalanK.24263171Biochim, Biophys. Acta20141842154], here we show that phosphorylation of PAGE4 weakens its interaction with c-Jun in vitro. These data suggest that phosphorylation induces conformational changes in natively disordered PAGE4 resulting in its decreased affinity for c-Jun to promote interaction of c-Jun with another, unidentified, partner. Alternatively, phosphorylated PAGE4 may induce transcription of a novel partner, which then potentiates c-Jun transactivation. Regardless, the present results clearly implicate PAGE4 as a component of the stress-response pathway and uncover a novel link between components of this pathway and prostatic development and disease.

The Cancer/Testis Antigen (CTA), Prostate-associated Gene 4 (PAGE4), is a stress-response protein that is upregulated in prostate cancer (PCa) especially in precursor lesions that result from inflammatory stress. In cells under stress, translocation of PAGE4 to mitochondria increases while production of reactive oxygen species decreases. Furthermore, PAGE4 is also upregulated in human fetal prostate, underscoring its potential role in development. However, the proteins that interact with PAGE4 and the mechanisms underlying its pleiotropic functions in prostatic development and disease remain unknown. Here, we identified c-Jun as a PAGE4 interacting partner. We show that both PAGE4 and c-Jun are overexpressed in the human fetal prostate; and in cell-based assays, PAGE4 robustly potentiates c-Jun transactivation. Single-molecule Förster resonance energy transfer experiments indicate that upon binding to c-Jun, PAGE4 undergoes conformational changes. However, no interaction is observed in presence of BSA or unilamellar vesicles containing the mitochondrial inner membrane diphosphatidylglycerol lipid marker cardiolipin. Together, our data indicate that PAGE4 specifically interacts with c-Jun and that, conformational dynamics may account for its observed pleiotropic functions. To our knowledge, this is the first report demonstrating crosstalk between a CTA and a proto-oncogene. Disrupting PAGE4/c-Jun interactions using small molecules may represent a novel therapeutic strategy for PCa.

Hypermutation and class switch recombination of immunoglobulin genes are antigen-activated mechanisms triggered by AID, a cytidine deaminase. AID deaminates cytidine residues in the DNA of the variable and the switch regions of the immunoglobulin locus. The resulting uracil induces error-prone DNA synthesis in the case of hypermutation or DNA breaks that activate nonhomologous recombination in the case of class-switch recombination. In vitro studies have demonstrated that AID deaminates single-stranded but not double-stranded substrates unless AID is in a complex with RPA and the substrate is actively undergoing transcription. However, it is not clear whether AID deaminates its substrates primarily as a monomer or as a higher order oligomer. To examine the oligomerization state of AID alone and in the presence of single stranded DNA substrates of various structures, including loops embedded in double-stranded DNA, we used atomic force microscopy (AFM) to visualize AID protein alone or in complex with DNA. Surprisingly, AFM results indicate that most AID molecules exist as a monomer and that it binds single-stranded DNA substrates as a monomer at concentrations where efficient deamination of single-stranded DNA substrates occur. The rate of deamination, under conditions of excess and limiting protein, also imply that AID can deaminate single-stranded substrates as a monomer. These results imply that nonphosphorylated AID is catalytically active as a monomer on single stranded DNA in vitro, including single-stranded DNA found in loops similar to those transiently formed in the immunoglobulin switch regions during transcription.

Background:The DNA repair factor MRE11-RAD50 is a multifunctional DNA complex with nuclease, DNA binding, and ATPase activity. Results: MRE11-RAD50 cleaves 3Ј-phosphotyrosyl bonds, such as those in trapped topoisomerase 1-DNA complexes, and then resects, generating a DNA end that can prime repair synthesis. Conclusion: MRE11-RAD50 is involved in the repair of trapped topoisomerase 1-DNA complexes. Significance: MRE11-RAD50 may be a useful biomarker for tumor sensitivity to topoisomerase 1 poisons.