RPA guides UNG to uracil in ssDNA to facilitate antibody class switching and repair of mutagenic uracil at the replication fork.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

RPA2 winged-helix domain facilitates UNG-mediated removal of uracil from ssDNA; implications for repair of mutagenic uracil at the replication fork.

Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2-3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential 'pre-replicative' removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.

MicroRNAs contribute to postnatal development of laminar differences and neuronal subtypes in the rat medial entorhinal cortex.

The medial entorhinal cortex (MEC) is important in spatial navigation and memory formation and its layers have distinct neuronal subtypes, connectivity, spatial properties, and disease susceptibility. As little is known about the molecular basis for the development of these laminar differences, we analyzed microRNA (miRNA) and messenger RNA (mRNA) expression differences between rat MEC layer II and layers III-VI during postnatal development. We identified layer and age-specific regulation of gene expression by miRNAs, which included processes related to neuron specialization and locomotor behavior. Further analyses by retrograde labeling and expression profiling of layer II stellate neurons and in situ hybridization revealed that the miRNA most up-regulated in layer II, miR-143, was enriched in stellate neurons, whereas the miRNA most up-regulated in deep layers, miR-219-5p, was expressed in ependymal cells, oligodendrocytes and glia. Bioinformatics analyses of predicted mRNA targets with negatively correlated expression patterns to miR-143 found that miR-143 likely regulates the Lmo4 gene, which is known to influence hippocampal-based spatial learning.

The proportion of CD16(+)CD14(dim) monocytes increases with tumor cell load in bone marrow of patients with multiple myeloma.

Multiple myeloma is an incurable cancer with expansion of malignant plasma cells in the bone marrow. Previous studies have shown that monocytes and macrophages in the bone marrow milieu are important for tumor growth and may play a role in the drug response. We therefore characterized monocytes in bone marrow aspirates by flow cytometry. We found that there was significant correlation between the proportion of CX3CR1 (+), CD16(+)CD14(dim) non classical monocytes, and percent plasma cells (PC) in the bone marrow of myeloma patients. The bone marrow monocytes could be stimulated by TLR ligands to produce cytokines which promote myeloma cell growth. The proportion of the non-classical monocytes increased with the tumor load, particularly in patients with tumor loads in the range of 10-30% bone marrow PC.

Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors.

Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation.

An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells.

Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.

A combined nuclear and nucleolar localization motif in activation-induced cytidine deaminase (AID) controls immunoglobulin class switching.

Activation-induced cytidine deaminase (AID) is a DNA mutator enzyme essential for adaptive immunity. AID initiates somatic hypermutation and class switch recombination (CSR) by deaminating cytosine to uracil in specific immunoglobulin (Ig) gene regions. However, other loci, including cancer-related genes, are also targeted. Thus, tight regulation of AID is crucial to balance immunity versus disease such as cancer. AID is regulated by several mechanisms including nucleocytoplasmic shuttling. Here we have studied nuclear import kinetics and subnuclear trafficking of AID in live cells and characterized in detail its nuclear localization signal. Importantly, we find that the nuclear localization signal motif also directs AID to nucleoli where it colocalizes with its interaction partner, catenin-ß-like 1 (CTNNBL1), and physically associates with nucleolin and nucleophosmin. Moreover, we demonstrate that release of AID from nucleoli is dependent on its C-terminal motif. Finally, we find that CSR efficiency correlates strongly with the arithmetic product of AID nuclear import rate and DNA deamination activity. Our findings suggest that directional nucleolar transit is important for the physiological function of AID and demonstrate that nuclear/nucleolar import and DNA cytosine deamination together define the biological activity of AID. This is the first study on subnuclear trafficking of AID and demonstrates a new level in its complex regulation. In addition, our results resolve the problem related to dissociation of deamination activity and CSR activity of AID mutants.

Nucleotide excision repair is associated with the replisome and its efficiency depends on a direct interaction between XPA and PCNA.

Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA(-/-) cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6-4) photoproducts, and are consequently more arrested in S phase as compared to XPA(-/-) cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.

Identification of a novel, widespread, and functionally important PCNA-binding motif.

Numerous proteins, many essential for the DNA replication machinery, interact with proliferating cell nuclear antigen (PCNA) through the PCNA-interacting peptide (PIP) sequence called the PIP box. We have previously shown that the oxidative demethylase human AlkB homologue 2 (hABH2) colocalizes with PCNA in replication foci. In this study, we show that hABH2 interacts with a posttranslationally modified PCNA via a novel PCNA-interacting motif, which we term AlkB homologue 2 PCNA-interacting motif (APIM). We identify APIM in >200 other proteins involved in DNA maintenance, transcription, and cell cycle regulation, and verify a functional APIM in five of these. Expression of an APIM peptide increases the cellular sensitivity to several cytostatic agents not accounted for by perturbing only the hABH2-PCNA interaction. Thus, APIM is likely to mediate PCNA binding in many proteins involved in DNA repair and cell cycle control during genotoxic stress.

Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA.

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells.

B cells from hyper-IgM patients carrying UNG mutations lack ability to remove uracil from ssDNA and have elevated genomic uracil.

The generation of high-affinity antibodies requires somatic hypermutation (SHM) and class switch recombination (CSR) at the immunoglobulin (Ig) locus. Both processes are triggered by activation-induced cytidine deaminase (AID) and require UNG-encoded uracil-DNA glycosylase. AID has been suggested to function as an mRNA editing deaminase or as a single-strand DNA deaminase. In the latter model, SHM may result from replicative incorporation of dAMP opposite U or from error-prone repair of U, whereas CSR may be triggered by strand breaks at abasic sites. Here, we demonstrate that extracts of UNG-proficient human B cell lines efficiently remove U from single-stranded DNA. In B cell lines from hyper-IgM patients carrying UNG mutations, the single-strand-specific uracil-DNA glycosylase, SMUG1, cannot complement this function. Moreover, the UNG mutations lead to increased accumulation of genomic uracil. One mutation results in an F251S substitution in the UNG catalytic domain. Although this UNG form was fully active and stable when expressed in Escherichia coli, it was mistargeted to mitochondria and degraded in mammalian cells. Our results may explain why SMUG1 cannot compensate the UNG2 deficiency in human B cells, and are fully consistent with the DNA deamination model that requires active nuclear UNG2. Based on our findings and recent information in the literature, we present an integrated model for the initiating steps in CSR.

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells.

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLbeta, POLdelta, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLbeta, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLbeta dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A).