Molecular dependencies and genomic consequences of a global DNA damage tolerance defect.

Background: DNA damage tolerance (DDT) enables replication to continue in the presence of fork stalling lesions. In mammalian cells, DDT is regulated by two independent pathways, controlled by the polymerase REV1 and ubiquitinated PCNA, respectively.

Results: To determine the molecular and genomic impact of a global DDT defect, we studied Pcna;Rev1 compound mutants in mouse cells.

Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair.

The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A but not mutation renders mammals hypersensitive to ICLs.

A C57BL/6J Fancg-KO Mouse Model Generated by CRISPR/Cas9 Partially Captures the Human Phenotype.

Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype.

Huwe1 supports B-cell development, B-cell-dependent immunity, somatic hypermutation and class switch recombination by regulating proliferation.

The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene () repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins.

Division of labor within the DNA damage tolerance system reveals non-epistatic and clinically actionable targets for precision cancer medicine.

Crosslink repair depends on the Fanconi anemia pathway and translesion synthesis polymerases that replicate over unhooked crosslinks. Translesion synthesis is regulated via ubiquitination of PCNA, and independently via translesion synthesis polymerase REV1. The division of labor between PCNA-ubiquitination and REV1 in interstrand crosslink repair is unclear.

The Widely Used Antihelmintic Drug Albendazole is a Potent Inducer of Loss of Heterozygosity.

The antihelmintic drug ABZ and its metabolites belong to the chemical family of benzimidazoles (BZM) that act as potent tubulin polymerization inhibitors, suggesting a potential re-direction of BZMs for cancer therapy. Applying UV-Vis spectrometry we here demonstrate ABZ as a DNA intercalator. This insight led us to determine the primary mode of ABZ action in mammalian cells.

The Ig heavy chain protein but not its message controls early B cell development.

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive allele is expressed, a phenomenon known as allelic exclusion. In contrast to a productively rearranged allele, the messenger RNA (mRNA) () from a nonproductively rearranged allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable to the molecular and developmental changes associated with the IgHCC.

Precision cancer therapy: profiting from tumor specific defects in the DNA damage tolerance system.

DNA damage tolerance (DDT) enables replication to continue in the presence of a damaged template and constitutes a key step in DNA interstrand crosslink repair. In this way DDT minimizes replication stress inflicted by a wide range of endogenous and exogenous agents, and provides a critical first line defense against alkylating and platinating chemotherapeutics. Effective DDT strongly depends on damage-induced, site-specific PCNA-ubiquitination at Lysine (K) 164 by the E2/E3 complex (RAD6/18).

PrimPol prevents APOBEC/AID family mediated DNA mutagenesis.

PrimPol is a DNA damage tolerant polymerase displaying both translesion synthesis (TLS) and (re)-priming properties. This led us to study the consequences of a PrimPol deficiency in tolerating mutagenic lesions induced by members of the APOBEC/AID family of cytosine deaminases. Interestingly, during somatic hypermutation, PrimPol counteracts the generation of C>G transversions on the leading strand.

Quantitative analysis by next generation sequencing of hematopoietic stem and progenitor cells (LSK) and of splenic B cells transcriptomes from wild-type and Usp3-knockout mice.

The data described here provide genome-wide expression profiles of murine primitive hematopoietic stem and progenitor cells (LSK) and of B cell populations, obtained by high throughput sequencing. Cells are derived from wild-type mice and from mice deficient for the ubiquitin-specific protease 3 (USP3; Usp3Δ/Δ). Modification of histone proteins by ubiquitin plays a crucial role in the cellular response to DNA damage (DDR) (Jackson and Durocher, 2013) [1].

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required.

Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells.

Histone ubiquitination at DNA breaks is required for activation of the DNA damage response (DDR) and DNA repair. How the dynamic removal of this modification by deubiquitinating enzymes (DUBs) impacts genome maintenance in vivo is largely unknown. To address this question, we generated mice deficient for Ub-specific protease 3 (USP3; Usp3Δ/Δ), a histone H2A DUB which negatively regulates ubiquitin-dependent DDR signaling.

Differential programming of B cells in AID deficient mice.

The Aicda locus encodes the activation induced cytidine deaminase (AID) and is highly expressed in germinal center (GC) B cells to initiate somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes. Besides these Ig specific activities in B cells, AID has been implicated in active DNA demethylation in non-B cell systems. We here determined a potential role of AID as an epigenetic eraser and transcriptional regulator in B cells.

Rev1 is essential in generating G to C transversions downstream of the Ung2 pathway but not the Msh2+Ung2 hybrid pathway.

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes are initiated by the enzymatic deamination of cytosine (C) to uracil (U). Uracil-DNA-glycosylase (Ung2) converts uracils into apyrimidinic (AP) sites, which is essential for the generation of transversions (TVs) at G/C basepairs during SHM and for efficient DNA break formation during CSR. Besides Ung2, the mismatch repair protein Msh2 and the translesion synthesis (TLS) DNA polymerase (Pol) Rev1 are implicated in SHM and CSR.

PCNA ubiquitination-independent activation of polymerase η during somatic hypermutation and DNA damage tolerance.

The generation of high affinity antibodies in B cells critically depends on translesion synthesis (TLS) polymerases that introduce mutations into immunoglobulin genes during somatic hypermutation (SHM). The majority of mutations at A/T base pairs during SHM require ubiquitination of PCNA at lysine 164 (PCNA-Ub), which activates TLS polymerases. By comparing the mutation spectra in B cells of WT, TLS polymerase η (Polη)-deficient, PCNA(K164R)-mutant, and PCNA(K164R);Polη double-mutant mice, we now find that most PCNA-Ub-independent A/T mutagenesis during SHM is mediated by Polη.

Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA.

Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164)) is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS) polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner.

HLTF and SHPRH are not essential for PCNA polyubiquitination, survival and somatic hypermutation: existence of an alternative E3 ligase.

DNA damage tolerance is regulated at least in part at the level of proliferating cell nuclear antigen (PCNA) ubiquitination. Monoubiquitination (PCNA-Ub) at lysine residue 164 (K164) stimulates error-prone translesion synthesis (TLS), Rad5-dependent polyubiquitination (PCNA-Ub(n)) stimulates error-free template switching (TS). To generate high affinity antibodies by somatic hypermutation (SHM), B cells profit from error-prone TLS polymerases.

The Fanconi anemia core complex is dispensable during somatic hypermutation and class switch recombination.

To generate high affinity antibodies during an immune response, B cells undergo somatic hypermutation (SHM) of their immunoglobulin genes. Error-prone translesion synthesis (TLS) DNA polymerases have been reported to be responsible for all mutations at template A/T and at least a fraction of G/C transversions. In contrast to A/T mutations which depend on PCNA ubiquitination, it remains unclear how G/C transversions are regulated during SHM.

Dependence of nucleotide substitutions on Ung2, Msh2, and PCNA-Ub during somatic hypermutation.

During somatic hypermutation (SHM), B cells introduce mutations into their immunoglobulin genes to generate high affinity antibodies. Current models suggest a separation in the generation of G/C transversions by the Ung2-dependent pathway and the generation of A/T mutations by the Msh2/ubiquitinated proliferating cell nuclear antigen (PCNA-Ub)-dependent pathway. It is currently unknown whether these pathways compete to initiate mutagenesis and whether PCNA-Ub functions downstream of Ung2.

Somatic hypermutation of immunoglobulin genes: lessons from proliferating cell nuclear antigenK164R mutant mice.

Proliferating cell nuclear antigen (PCNA) encircles DNA as a ring-shaped homotrimer and, by tethering DNA polymerases to their template, PCNA serves as a critical replication factor. In contrast to high-fidelity DNA polymerases, the activation of low-fidelity translesion synthesis (TLS) DNA polymerases seems to require damage-inducible monoubiquitylation (Ub) of PCNA at lysine residue 164 (PCNA-Ub). TLS polymerases can tolerate DNA damage, i.

A/T mutagenesis in hypermutated immunoglobulin genes strongly depends on PCNAK164 modification.

B cells use translesion DNA synthesis (TLS) to introduce somatic mutations around genetic lesions caused by activation-induced cytidine deaminase. Monoubiquitination at lysine(164) of proliferating cell nuclear antigen (PCNA(K164)) stimulates TLS. To determine the role of PCNA(K164) modifications in somatic hypermutation, PCNA(K164R) knock-in mice were generated.

Immunogenicity, including vitiligo, and feasibility of vaccination with autologous GM-CSF-transduced tumor cells in metastatic melanoma patients.

Purpose: To determine the feasibility, toxicity, and immunologic effects of vaccination with autologous tumor cells retrovirally transduced with the GM-CSF gene, we performed a phase I/II vaccination study in stage IV metastatic melanoma patients.

Patients And Methods: Sixty-four patients were randomly assigned to receive three vaccinations of high-dose or low-dose tumor cells at 3-week intervals. Tumor cell vaccine preparation succeeded for 56 patients (88%), but because of progressive disease, the well-tolerated vaccination was completed in only 28 patients.