A hybrid mechanism of action for BCL6 in B cells defined by formation of functionally distinct complexes at enhancers and promoters.

The BCL6 transcriptional repressor is required for the development of germinal center (GC) B cells and diffuse large B cell lymphomas (DLBCLs). Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B cells is unknown. We find that in B cells, BCL6 mostly functions through two independent mechanisms that are collectively essential to GC formation and DLBCL, both mediated through its N-terminal BTB domain. These are (1) the formation of a unique ternary BCOR-SMRT complex at promoters, with each corepressor binding to symmetrical sites on BCL6 homodimers linked to specific epigenetic chromatin features, and (2) the "toggling" of active enhancers to a poised but not erased conformation through SMRT-dependent H3K27 deacetylation, which is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Dynamic toggling of enhancers provides a basis for B cells to undergo rapid transcriptional and phenotypic changes in response to signaling or environmental cues.

Structural mechanism of replication stalling on a bulky amino-polycyclic aromatic hydrocarbon DNA adduct by a y family DNA polymerase.

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties.

Systemic ceramide accumulation leads to severe and varied pathological consequences.

Farber disease (FD) is a severe inherited disorder of lipid metabolism characterized by deficient lysosomal acid ceramidase (ACDase) activity, resulting in ceramide accumulation. Ceramide and metabolites have roles in cell apoptosis and proliferation. We introduced a single-nucleotide mutation identified in human FD patients into the murine Asah1 gene to generate the first model of systemic ACDase deficiency. Homozygous Asah1(P361R/P361R) animals showed ACDase defects, accumulated ceramide, demonstrated FD manifestations and died within 7-13 weeks. Mechanistically, MCP-1 levels were increased and tissues were replete with lipid-laden macrophages. Treatment of neonates with a single injection of human ACDase-encoding lentivector diminished the severity of the disease as highlighted by enhanced growth, decreased ceramide, lessened cellular infiltrations and increased lifespans. This model of ACDase deficiency offers insights into the pathophysiology of FD and the roles of ACDase, ceramide and related sphingolipids in cell signaling and growth, as well as facilitates the development of therapy.

Replication of a carcinogenic nitropyrene DNA lesion by human Y-family DNA polymerase.

Nitrated polycyclic aromatic hydrocarbons are common environmental pollutants, of which many are mutagenic and carcinogenic. 1-Nitropyrene is the most abundant nitrated polycyclic aromatic hydrocarbon, which causes DNA damage and is carcinogenic in experimental animals. Error-prone translesion synthesis of 1-nitropyrene-derived DNA lesions generates mutations that likely play a role in the etiology of cancer. Here, we report two crystal structures of the human Y-family DNA polymerase iota complexed with the major 1-nitropyrene DNA lesion at the insertion stage, incorporating either dCTP or dATP nucleotide opposite the lesion. Polι maintains the adduct in its active site in two distinct conformations. dCTP forms a Watson-Crick base pair with the adducted guanine and excludes the pyrene ring from the helical DNA, which inhibits replication beyond the lesion. By contrast, the mismatched dATP stacks above the pyrene ring that is intercalated in the helix and achieves a productive conformation for misincorporation. The intra-helical bulky pyrene mimics a base pair in the active site and facilitates adenine misincorporation. By structure-based mutagenesis, we show that the restrictive active site of human polη prevents the intra-helical conformation and A-base misinsertions. This work provides one of the molecular mechanisms for G to T transversions, a signature mutation in human lung cancer.

Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase.

The ability of DNA polymerases to differentiate between ribonucleotides and deoxribonucleotides is fundamental to the accurate replication and maintenance of an organism's genome. The active sites of Y-family DNA polymerases are highly solvent accessible, yet these enzymes still maintain a high selectivity towards deoxyribonucleotides. Here, we biochemically demonstrate that a single active-site mutation (Y12A) in Dpo4, a model Y-family DNA polymerase, causes both a dramatic loss of ribonucleotide discrimination and a decrease in nucleotide incorporation efficiency. We also determined two ternary crystal structures of the Dpo4 Y12A mutant incorporating either dATP or ATP nucleotides opposite a template dT base. Interestingly, both dATP and ATP were hydrolyzed to dADP and ADP, respectively. In addition, the dADP and ADP molecules adopt a similar conformation and position at the polymerase active site to a ddADP molecule in the ternary crystal structure of wild-type Dpo4. The Y12A mutant loses stacking interactions with the deoxyribose of dNTP, which destabilizes the binding of incoming nucleotides. The mutation also opens a space to accommodate the 2'-OH group of the ribose of NTP in the polymerase active site. The structural change leads to the reduction in deoxynucleotide incorporation efficiency and allows ribonucleotide incorporation.

Unique active site promotes error-free replication opposite an 8-oxo-guanine lesion by human DNA polymerase iota.

The 8-oxo-guanine (8-oxo-G) lesion is the most abundant and mutagenic oxidative DNA damage existing in the genome. Due to its dual coding nature, 8-oxo-G causes most DNA polymerases to misincorporate adenine. Human Y-family DNA polymerase iota (polι) preferentially incorporates the correct cytosine nucleotide opposite 8-oxo-G. This unique specificity may contribute to polι's biological role in cellular protection against oxidative stress. However, the structural basis of this preferential cytosine incorporation is currently unknown. Here we present four crystal structures of polι in complex with DNA containing an 8-oxo-G lesion, paired with correct dCTP or incorrect dATP, dGTP, and dTTP nucleotides. An exceptionally narrow polι active site restricts the purine bases in a syn conformation, which prevents the dual coding properties of 8-oxo-G by inhibiting syn/anti conformational equilibrium. More importantly, the 8-oxo-G base in a syn conformation is not mutagenic in polι because its Hoogsteen edge does not form a stable base pair with dATP in the narrow active site. Instead, the syn 8-oxo-G template base forms the most stable replicating base pair with correct dCTP due to its small pyrimidine base size and enhanced hydrogen bonding with the Hoogsteen edge of 8-oxo-G. In combination with site directed mutagenesis, we show that Gln59 in the finger domain specifically interacts with the additional O(8) atom of the lesion base, which influences nucleotide selection, enzymatic efficiency, and replication stalling at the lesion site. Our work provides the structural mechanism of high-fidelity 8-oxo-G replication by a human DNA polymerase.

Structural basis of error-prone replication and stalling at a thymine base by human DNA polymerase iota.

Human DNA polymerase iota (pol iota) is a unique member of Y-family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of pol complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson-Crick base pairing by pol. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H-bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template 'U-turn' is stabilized by pol and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain-swapping experiments indicate that the finger domain is responsible for pol's high error rates on pyrimidines and determines the incorporation specificity.

Structural insight into recruitment of translesion DNA polymerase Dpo4 to sliding clamp PCNA.

DNA polymerases are co-ordinated by sliding clamps (PCNA/beta-clamp) in translesion synthesis. It is unclear how these enzymes assemble on PCNA with geometric and functional compatibility. We report the crystal structure of a full-length Y-family polymerase, Dpo4, in complex with heterodimeric PCNA1-PCNA2 at 2.05 A resolution. Dpo4 exhibits an extended conformation that differs from the Dpo4 structures in apo- or DNA-bound form. Two hinges have been identified in Dpo4, which render the multidomain polymerase flexible conformations and orientations relative to PCNA. Dpo4 binds specifically to PCNA1 on the conserved ligand binding site. The C-terminal peptide of Dpo4 becomes structured with a 3(10) helix and dominates the specific binding. The Y-family polymerase also contacts PCNA1 with its finger, thumb and little finger domains, which are conformation-dependent protein-protein interactions that diversify the binding mode of Dpo4 on PCNA. The structure reveals a molecular model in which substrate/partner binding-coupled multiple conformations of a Y-family polymerase facilitate its recruitment and co-ordination on the sliding clamp. The conformational flexibility would turn the error-prone Y-family polymerase off when more efficient high-fidelity DNA polymerases work on undamaged DNA and turn it onto DNA templates to perform translesion synthesis when replication forks are stalled by DNA lesions.