For the Better or for the Worse? The Effect of Manganese on the Activity of Eukaryotic DNA Polymerases.

DNA polymerases constitute a versatile group of enzymes that not only perform the essential task of genome duplication but also participate in various genome maintenance pathways, such as base and nucleotide excision repair, non-homologous end-joining, homologous recombination, and translesion synthesis. Polymerases catalyze DNA synthesis via the stepwise addition of deoxynucleoside monophosphates to the 3' primer end in a partially double-stranded DNA. They require divalent metal cations coordinated by active site residues of the polymerase. Mg2+ is considered the likely physiological activator because of its high cellular concentration and ability to activate DNA polymerases universally. Mn2+ can also activate the known DNA polymerases, but in most cases, it causes a significant decrease in fidelity and/or processivity. Hence, Mn2+ has been considered mutagenic and irrelevant during normal cellular function. Intriguingly, a growing body of evidence indicates that Mn2+ can positively influence some DNA polymerases by conferring translesion synthesis activity or altering the substrate specificity. Here, we review the relevant literature focusing on the impact of Mn2+ on the biochemical activity of a selected set of polymerases, namely, Polß, Polλ, and Polµ, of the X family, as well as Polι and Polη of the Y family of polymerases, where congruous data implicate the physiological relevance of Mn2+ in the cellular function of these enzymes.

Manganese Is a Strong Specific Activator of the RNA Synthetic Activity of Human Polη.

DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.

Selective Metal Ion Utilization Contributes to the Transformation of the Activity of Yeast Polymerase η from DNA Polymerization toward RNA Polymerization.

Polymerase eta (Polη) is a translesion synthesis DNA polymerase directly linked to cancer development. It can bypass several DNA lesions thereby rescuing DNA damage-stalled replication complexes. We previously presented evidence implicating Saccharomyces cerevisiae Polη in transcription elongation, and identified its specific RNA extension and translesion RNA synthetic activities. However, RNA synthesis by Polη proved rather inefficient under conditions optimal for DNA synthesis. Searching for factors that could enhance its RNA synthetic activity, we have identified the divalent cation of manganese. Here, we show that manganese triggers drastic changes in the activity of Polη. Kinetics experiments indicate that manganese increases the efficiency of ribonucleoside incorporation into RNA by ~400-2000-fold opposite undamaged DNA, and ~3000 and ~6000-fold opposite TT dimer and 8oxoG, respectively. Importantly, preference for the correct base is maintained with manganese during RNA synthesis. In contrast, activity is strongly impaired, and base discrimination is almost lost during DNA synthesis by Polη with manganese. Moreover, Polη shows strong preference for manganese during RNA synthesis even at a 25-fold excess magnesium concentration. Based on this, we suggest that a new regulatory mechanism, selective metal cofactor utilization, modulates the specificity of Polη helping it to perform distinct activities needed for its separate functions during replication and transcription.

Dominant suppressor genes of p53-induced apoptosis in Drosophila melanogaster.

One of a major function of programmed cell death (apoptosis) is the removal of cells which suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes which, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS-promoters at both ends which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS-promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, seven genes were identified which suppressed the p53-induced apoptosis. In four mutants, the suppression effect resulted from single genes activated by one UAS-promoter (Pka-R2, Rga, crol, Spt5). In the other three (Orct2, Polr2M, stg), deleting either UAS-promoter eliminated the suppression effect. In qPCR experiments we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eucaryotic genomes there are co-expressed gene clusters. Three of the DEP insertion mutants are included and two are in close vicinity of separate co-expressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.

Elevated level of lysine 9-acetylated histone H3 at the MDR1 promoter in multidrug-resistant cells.

Failure of chemotherapy in breast cancer presents a major problem and is often due to elevated expression of ATP binding cassette (ABC)-type transporters, such as MDR1 protein. It has been shown that MDR1/ABCB1 gene expression is regulated at the chromatin level by DNA methylation and histone acetylation. However, the modified histone residues have not been identified and the role of various histone acetyl transferases (HATs) is not fully understood. By studying a breast carcinoma model cell line and its MDR1-overexpressing derivative, we show that the histone 3 lysine 9 (H3K9) acetylation level is elevated 100-fold in the promoter and first exon of the MDR1 gene in the drug-resistant cell line compared to the drug-sensitive cell line. The acetylation level of the other examined lysine residues (H3K4, H3K14, H4K8, and H4K12) is weakly or not at all elevated in the MDR1 locus, although their acetylation is generally increased genome-wide in the drug-resistant cell. Downregulation of the expression of HATs PCAF and GCN5 by RNAi effectively reduces the expression of MDR1. Unexpectedly, treatment with a p300-selective inhibitor (HAT inhibitor II) further increases MDR1 expression and drug efflux in the drug-resistant cells. Our data suggest that repeated exposure to chemotherapy may result in deregulated histone acetylation genome-wide and in the MDR1 promoter.

The inner side of yeast PCNA contributes to genome stability by mediating interactions with Rad18 and the replicative DNA polymerase δ.

PCNA is a central orchestrator of cellular processes linked to DNA metabolism. It is a binding platform for a plethora of proteins and coordinates and regulates the activity of several pathways. The outer side of PCNA comprises most of the known interacting and regulatory surfaces, whereas the residues at the inner side constitute the sliding surface facing the DNA double helix. Here, by investigating the L154A mutation found at the inner side, we show that the inner surface mediates protein interactions essential for genome stability. It forms part of the binding site of Rad18, a key regulator of DNA damage tolerance, and is required for PCNA sumoylation which prevents unscheduled recombination during replication. In addition, the L154 residue is necessary for stable complex formation between PCNA and the replicative DNA polymerase δ. Hence, its absence increases the mutation burden of yeast cells due to faulty replication. In summary, the essential role of the L154 of PCNA in guarding and maintaining stable replication and promoting DNA damage tolerance reveals a new connection between these processes and assigns a new coordinating function to the central channel of PCNA.

Protein array based interactome analysis of amyloid-ß indicates an inhibition of protein translation.

Oligomeric amyloid-ß is currently of interest in amyloid-ß mediated toxicity and the pathogenesis of Alzheimer's disease. Mapping the amyloid-ß interaction partners could help to discover novel pathways in disease pathogenesis. To discover the amyloid-ß interaction partners, we applied a protein array with more than 8100 unique recombinantly expressed human proteins. We identified 324 proteins as potential interactors of oligomeric amyloid-ß. The Gene Ontology functional analysis of these proteins showed that oligomeric amyloid-ß bound to multiple proteins with diverse functions both from extra and intracellular localizations. This undiscriminating binding phenotype indicates that multiple protein interactions mediate the toxicity of the oligomeric amyloid-ß. The most highly impacted cellular system was the protein translation machinery. Oligomeric amyloid-ß could bind to altogether 24 proteins involved in translation initiation and elongation. The binding of amyloid-ß to purified rat hippocampal ribosomes validated the protein array results. More importantly, in vitro translation assays showed that the oligomeric amyloid-ß had a concentration dependent inhibitory activity on translation. Our results indicate that the inhibited protein synthesis is one of the pathways that can be involved in the amyloid-beta induced neurotoxicity.

The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5.

DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

TATA binding protein associated factor 3 (TAF3) interacts with p53 and inhibits its function.

BACKGROUND: The tumour suppressor protein p53 is a sequence specific DNA-binding transcription regulator, which exerts its versatile roles in genome protection and apoptosis by affecting the expression of a large number of genes. In an attempt to obtain a better understanding of the mechanisms by which p53 transcription function is regulated, we studied p53 interactions. RESULTS: We identified BIP2 (Bric-à-brac interacting protein 2), the fly homolog of TAF3, a histone fold and a plant homeodomain containing subunit of TFIID, as an interacting partner of Drosophila melanogaster p53 (Dmp53). We detected physical interaction between the C terminus of Dmp53 and the central region of TAF3 both in yeast two hybrid assays and in vitro. Interestingly, DmTAF3 can also interact with human p53, and mammalian TAF3 can bind to both Dmp53 and human p53. This evolutionarily conserved interaction is functionally significant, since elevated TAF3 expression severely and selectively inhibits transcription activation by p53 in human cell lines, and it decreases the level of the p53 protein as well. CONCLUSION: We identified TAF3 as an evolutionarily conserved negative regulator of p53 transcription activation function.

Different sets of genes are activated by p53 upon UV or ionizing radiation in Drosophila melanogaster.

The p53 tumour suppressor plays central role in the maintenance of genome integrity. P53 deficient fruit flies are highly sensitive to ionizing radiation (IR) and show genome instability suggesting that the Drosophila melanogaster p53 (Dmp53) is necessary for the proper damage response upon IR. We found that Dmp53 null fruit flies are highly sensitive to ultraviolet radiation (UV) as well. We analyzed the expression levels of apoptotic genes in wild type and Dmp53 null mutant animals after UV or IR using quantitative real-time RT-PCR. Ark (Apaf-1 related killer) was induced in a Dmp53-dependent way upon UV treatment but not by IR, hid (head involution defective/wrinkled) was induced upon both types of DNA damage, while reaper was induced only upon IR but not UV treatment. Using microarray analysis we identified several further genes that are activated upon UV irradiation in the presence of wild type Dmp53 only. Some but not all of these genes show Dmp53-dependent activation upon IR treatment as well. These results suggest that Dmp53 activates distinct cellular pathways through regulation of different target genes after different types of DNA damage.

Translesion synthesis DNA polymerase η exhibits a specific RNA extension activity and a transcription-associated function.

Polymerase eta (Polη) is a low fidelity translesion synthesis DNA polymerase that rescues damage-stalled replication by inserting deoxy-ribonucleotides opposite DNA damage sites resulting in error-free or mutagenic damage bypass. In this study we identify a new specific RNA extension activity of Polη of Saccharomyces cerevisiae. We show that Polη is able to extend RNA primers in the presence of ribonucleotides (rNTPs), and that these reactions are an order of magnitude more efficient than the misinsertion of rNTPs into DNA. Moreover, during RNA extension Polη performs error-free bypass of the 8-oxoguanine and thymine dimer DNA lesions, though with a 103 and 102-fold lower efficiency, respectively, than it synthesizes opposite undamaged nucleotides. Furthermore, in vivo experiments demonstrate that the transcription of several genes is affected by the lack of Polη, and that Polη is enriched over actively transcribed regions. Moreover, inactivation of its polymerase activity causes similar transcription inhibition as the absence of Polη. In summary, these results suggest that the new RNA synthetic activity of Polη can have in vivo relevance.

Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain.

Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well.

Daxx-like protein of Drosophila interacts with Dmp53 and affects longevity and Ark mRNA level.

Daxx-like protein (DLP), the Drosophila homolog of Daxx, binds Drosophila melanogaster p53 (Dmp53) through its C-terminal region. We generated DLP mutants and found that although DLP expression is developmentally regulated, it is not essential for the execution of the developmental program. The effects DLP mutations show in the loss of heterozygosity assay and on phenotypes resulting from Dmp53 overexpression indicate a genetic interaction between DLP and Dmp53. In contrast to Dmp53 mutants, however, loss of DLP does not result in radiosensitivity indicating that it does not play an essential role in the activation of Dmp53-dependent response after ionizing radiation, and DLP is also not required for the irradiation-induced activation of reaper. In contrast, DLP is involved in the transcriptional regulation of Ark, because Ark mRNA level is decreased in DLP mutants and increased upon ectopic overexpression of DLP. Interestingly, DLP mutants have reduced longevity and reduced female fertility. Altogether, our data suggest complex functions for DLP, which include an anti-apoptotic effect exerted through repression of some Dmp53 functions, and activation of some proapoptotic genes.

Induction of p57(KIP2) expression by p73beta.

The p53-related protein p73 has many functions similar to that of p53 including the ability to induce cell-cycle arrest and apoptosis. Both p53 and p73 function as transcription factors, and p73 activates expression of many genes that also are regulated by p53. Despite their similarities, it is evident that p53 and p73 are not interchangeable functionally, with p73 playing a role in normal growth and development that is not shared by p53. In this paper we describe the ability of p73beta but not p53 to activate expression of the cyclin-dependent kinase inhibitor p57(KIP) and KvLQT1, two genes that are coregulated in an imprinted region of the genome. Our results suggest that p73 may regulate expression of genes through mechanisms that are not shared by p53, potentially explaining the different contributions of p53 and p73 to normal development.

Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation.

Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP-2, a member of the TGFbeta superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP-mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP-2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP-2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP-2 and expression was modulated from 3- to 14-fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up- or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP-2 immediately responsive genes (1-4 h), which exhibited either transient or sustained expression, reflect activation and repression of non-osseous BMP-2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2-8 h). Commitment to the osteogenic phenotype is recognized by 8 h, reflected by downregulation of most myogenic-related genes and induction of a spectrum of signaling proteins and enzymes facilitating synthesis and assembly of an extracellular skeletal environment. These genes included collagens Type I and VI and the small leucine rich repeat family of proteoglycans (e.g., decorin, biglycan, osteomodulin, fibromodulin, and osteoadherin/osteoglycin) that reached peak expression at 24 h. With extracellular matrix development, the bone phenotype was further established from 16 to 24 h by induction of genes for cell adhesion and communication and enzymes that organize the bone ECM. Our microarray analysis resulted in the discovery of a class of genes, initially described in relation to differentiation of astrocytes and oligodendrocytes that are functionally coupled to signals for cellular extensions. They include nexin, neuropilin, latexin, neuroglian, neuron specific gene 1, and Ulip; suggesting novel roles for these genes in the bone microenvironment. This global analysis identified a multistage molecular and cellular cascade that supports BMP-2-mediated osteoblast differentiation.

Runx1/AML1 hematopoietic transcription factor contributes to skeletal development in vivo.

The requirement of Runx2 (Cbfal/AML3), a runt homology domain transcription factor essential for bone formation and osteoblast differentiation, is well established. Although Runx2 is expressed in the developing embryo prior to ossification, yet in the absence of Runx2 initial formation of the skeleton is normal, suggesting a potential redundancy in function of Runx family members. Here we addressed expression of the hematopoietic family member Runx1 (AML1/Cbfa2) in relation to skeletal development using a LacZ knock-in mouse model (Runx1(lz/+)). The resulting fusion protein reflects Runx1 promoter activity in its native context. Our studies show that Runx1 is expressed by prechondrocytic tissue forming the cartilaginous anlagen in the embryo, resting zone chondrocytes, suture lines of the calvarium, and in periosteal and perichondral membranes of all bone. Runx1 continues to be expressed in these tissues in adult mice, but is absent in mature cartilage or mineralized bone. However, hyaline cartilage outside the bone environment (trachea, xiphoid tissues), and epithelium of many soft tissues (trachea, thyroid, lung, skin) also express Runx1. The robust expression of Runx1 in vivo in chondroblasts at sites of cartilage growth and in osteoblasts at sites of new bone formation, suggests that Runx1 expression may be related to osteochondroprogenitor cell differentiation. This observation is further supported by high expression of Runx1 in ex vivo cultures of marrow stromal cells and calvarial derived osteoblasts from Runx1(lz/+) mice. These data indicate that Runx1 may contribute to the early stages of skeletogenesis and continues to function in the progenitor cells of tissues that support bone formation in the adult.