Replicative Polymerases Research Articles

A gatekeeping function of the replicative polymerase controls pathway choice in the resolution of lesion-stalled replisomes

DNA lesions stall the replisome and proper resolution of these obstructions is critical for genome stability. Replisomes can directly replicate past a lesion by error-prone translesion synthesis. Alternatively, replisomes can reprime DNA synthesis downstream of the lesion, creating a single-stranded DNA gap that is repaired primarily in an error-free, homology-directed manner. Here we demonstrate how structural changes within the Escherichia coli replisome determine the resolution pathway of lesion-stalled replisomes. This pathway selection is controlled by a dynamic interaction between the proofreading subunit of the replicative polymerase and the processivity clamp, which sets a kinetic barrier to restrict access of translesion synthesis (TLS) polymerases to the primer/template junction. Failure of TLS polymerases to overcome this barrier leads to repriming, which competes kinetically with TLS. Our results demonstrate that independent of its exonuclease activity, the proofreading subunit of the replisome acts as a gatekeeper and influences replication fidelity during the resolution of lesion-stalled replisomes.

Translesion synthesis DNA polymerases η, ι, and ν promote mutagenic replication through the anticancer nucleoside cytarabine

Cytarabine (AraC) is the mainstay for the treatment of acute myeloid leukemia. Although complete remission is observed in a large proportion of patients, relapse occurs in almost all the cases. The chemotherapeutic action of AraC derives from its ability to inhibit DNA synthesis by the replicative polymerases (Pols); the replicative Pols can insert AraCTP at the 3' terminus of the nascent DNA strand, but they are blocked at extending synthesis from AraC. By extending synthesis from the 3'-terminal AraC and by replicating through AraC that becomes incorporated into DNA, translesion synthesis (TLS) DNA Pols could reduce the effectiveness of AraC in chemotherapy. Here we identify the TLS Pols required for replicating through the AraC templating residue and determine their error-proneness. We provide evidence that TLS makes a consequential contribution to the replication of AraC-damaged DNA; that TLS through AraC is conducted by three different pathways dependent upon Polη, Polι, and Polν, respectively; and that TLS by all these Pols incurs considerable mutagenesis. The prominent role of TLS in promoting proficient and mutagenic replication through AraC suggests that TLS inhibition in acute myeloid leukemia patients would increase the effectiveness of AraC chemotherapy; and by reducing mutation formation, TLS inhibition may dampen the emergence of drug-resistant tumors and thereby the high incidence of relapse in AraC-treated patients.

High Tumor Mutation Burden and Other Immunotherapy Response Predictors in Breast Cancers: Associations and Therapeutic Opportunities

The recent development of effective immunotherapies with immune checkpoint inhibitors for the treatment of cancer has rekindled the interest for the immune system and its activation for an anti-cancer response. At the same time, it has become evident that not all types of cancers respond equally to these treatments, and even within the same tumor type only a subset of patients derive clinical benefit. Biomarkers predictive of response to immunotherapy have been sought and in certain occasions incorporated in the indication for treatment. These include expression of PD-L1 and defects in DNA mismatch repair (MMR). Tumor mutation burden (TMB) has been associated with response to immune checkpoint inhibitors. The current investigation examines TMB as a biomarker of response to immunotherapy in breast cancer. Publicly available data from the breast cancer study of The Cancer Genome Atlas (TCGA) and the METABRIC study were analyzed. Parameters examined included the TMB and specific mutations that may impact on TMB. In addition, correlations with breast cancer sub-types were investigated. The percentage of breast cancers with high TMB (more than 192 mutations per sample) was low (3.5-4.6%) in luminal and triple-negative cancers and higher (14.1%) in the HER2-positive subset. Almost all cancers with high TMB had defects in MMR proteins or the replicative polymerases POLE and POLD1. Small sub-sets of breast cancers with high TMB exist and may present an opportunity for effective immunotherapeutic targeting.

Structural insights into mutagenicity of anticancer nucleoside analog cytarabine during replication by DNA polymerase \u03b7

Cytarabine (AraC) is the mainstay chemotherapy for acute myeloid leukemia (AML). Whereas initial treatment with AraC is usually successful, most AML patients tend to relapse, and AraC treatment-induced mutagenesis may contribute to the development of chemo-resistant leukemic clones. We show here that whereas the high-fidelity replicative polymerase Polδ is blocked in the replication of AraC, the lower-fidelity translesion DNA synthesis (TLS) polymerase Polη is proficient, inserting both correct and incorrect nucleotides opposite a template AraC base. Furthermore, we present high-resolution crystal structures of human Polη with a template AraC residue positioned opposite correct (G) and incorrect (A) incoming deoxynucleotides. We show that Polη can accommodate local perturbation caused by the AraC via specific hydrogen bonding and maintain a reaction-ready active site alignment for insertion of both correct and incorrect incoming nucleotides. Taken together, the structures provide a novel basis for the ability of Polη to promote AraC induced mutagenesis in relapsed AML patients.

DNA polymerases in the risk and prognosis of colorectal and pancreatic cancers.

Human cancers arise from the alteration of genes involved in important pathways that mainly affect cell growth and proliferation. DNA replication and DNA damages recognition and repair are among these pathways and DNA polymerases that take part in these processes are frequently involved in cancer onset and progression. For example, damaging alterations within the proofreading domain of replicative polymerases, often reported in patients affected by colorectal cancer (CRC), are considered risk factors and drivers of carcinogenesis as they can lead to the accumulation of several mutations throughout the genome. Thus, replicative polymerases can be involved in cancer when losses of their physiological functions occur. On the contrary, reparative polymerases are often involved in cancer precisely because of their physiological role. In fact, their ability to repair and bypass DNA damages, which confers genome stability, can also counteract the effect of most anticancer drugs. In addition, the altered expression can characterise some type of cancers, which exacerbates this aspect. For example, all of the DNA polymerases involved a damage bypass mechanism, known as translesion synthesis, with the only exception of polymerase theta, are downregulated in CRC. Conversely, in pancreatic ductal adenocarcinoma (PDAC), most of these polymerase result upregulated. This suggests that different types of cancer can rely on different reparative polymerases to acquire drug resistance. Here we will examine all of the aspects that link DNA polymerases with CRC and PDAC.

Transcriptional Fidelity of Mitochondrial RNA Polymerase RpoTm from Arabidopsis thaliana

Fidelity of RNA synthesis is essential for the faithful transfer of information from DNA to RNA. A comprehensive analysis of the nucleotide selectivity by the mitochondrial RNA polymerase (RNAP) RpoTm, from Arabidopsis thaliana, has been carried out. The kinetic parameters for the incorporation of cognate, noncognate, and oxidized bases have been determined. The results establish high fidelity of mitochondrial transcription resembling those of replicative polymerases in the absence of repair. In addition, RpoTm incorporates oxidized nucleotides with similar efficiency compared with mismatches and is capable of extending the RNA beyond the insertion of the oxidized base. Furthermore, lesion bypass study on RpoTm demonstrates that the enzyme bypasses 8-oxo-guanine by insertion of adenine leading to C to A mutations in RNA. Homology modeling of RpoTm elongation complex allows delineation of the residues necessary for stabilizing the incoming NTP substrate and for posing the template nucleotide residue. Substitution of these residues leads to compromise in the activity of the enzyme corroborating their importance in RNA synthesis. Comparison of the data with T7 RNAPs indicates that low efficiency of misincorporation is a universal strategy used by single-subunit RNAPs for maintaining high fidelity in the absence of proofreading and repair activity in mitochondria.

Panicum Mosaic Virus and Its Satellites Acquire RNA Modifications Associated with Host-Mediated Antiviral Degradation.

Positive-sense RNA viruses in the Tombusviridae family have genomes lacking a 5' cap structure and prototypical 3' polyadenylation sequence. Instead, these viruses utilize an extensive network of intramolecular RNA-RNA interactions to direct viral replication and gene expression. Here we demonstrate that the genomic RNAs of Panicum mosaic virus (PMV) and its satellites undergo sequence modifications at their 3' ends upon infection of host cells. Changes to the viral and subviral genomes arise de novo within Brachypodium distachyon (herein called Brachypodium) and proso millet, two alternative hosts of PMV, and exist in the infections of a native host, St. Augustinegrass. These modifications are defined by polyadenylation [poly(A)] events and significant truncations of the helper virus 3' untranslated region-a region containing satellite RNA recombination motifs and conserved viral translational enhancer elements. The genomes of PMV and its satellite virus (SPMV) were reconstructed from multiple poly(A)-selected Brachypodium transcriptome data sets. Moreover, the polyadenylated forms of PMV and SPMV RNAs copurify with their respective mature icosahedral virions. The changes to viral and subviral genomes upon infection are discussed in the context of a previously understudied poly(A)-mediated antiviral RNA degradation pathway and the potential impact on virus evolution.IMPORTANCE The genomes of positive-sense RNA viruses have an intrinsic capacity to serve directly as mRNAs upon viral entry into a host cell. These RNAs often lack a 5' cap structure and 3' polyadenylation sequence, requiring unconventional strategies for cap-independent translation and subversion of the cellular RNA degradation machinery. For tombusviruses, critical translational regulatory elements are encoded within the 3' untranslated region of the viral genomes. Here we describe RNA modifications occurring within the genomes of Panicum mosaic virus (PMV), a prototypical tombusvirus, and its satellite agents (i.e., satellite virus and noncoding satellite RNAs), all of which depend on the PMV-encoded RNA polymerase for replication. The atypical RNAs are defined by terminal polyadenylation and truncation events within the 3' untranslated region of the PMV genome. These modifications are reminiscent of host-mediated RNA degradation strategies and likely represent a previously underappreciated defense mechanism against invasive nucleic acids.

A Comprehensive Superposition of Viral Polymerase Structures.

Nucleic acid polymerases are essential enzymes that replicate the genomes of both RNA and DNA viruses. These enzymes are generally encoded by viruses themselves so as to provide biochemical functions and control elements that differ from those of the host cell polymerases. The core active site structure used by all replicative polymerases is highly conserved and composed of two key aspartate residues from the conserved motifs A and C, but beyond this there is significant divergence among structures. These differences can make it difficult to select which portions of structures to align for comparisons, yet there are extended structural similarities within different groups of viral polymerases that should clearly be considered to generate optimal alignments. This manuscript describes a comprehensive structure-based superposition of every viral polymerase structure solved thus far based on an alignment-tree approach wherein aligned regions grow in complexity as similarity among polymerases increases. The result is a set of 646 structures that have been aligned into a single common orientation. This provides a convenient resource for directly comparing viral polymerases and illustrating structural conservation among them. It also sets the stage for detailed bioinformatics analysis to further assess common structural features. The full set of protein data bank (PDB) formatted files is publicly available via the Polymerase Structures community page at the Zenodo.org open data repository.

A61 Large RNA genomes: Is RNA polymerase fidelity enough?

Large-genome Nidoviruses and Nidovirus-like viruses reside at the current boundary of largest RNA genome sizes. They code for an unusually large number of gene products matching that of small DNA viruses (e.g. DNA bacteriophages). The order of appearance and distribution of enzyme genes along various virus families (e.g. helicase and ExoN) may be seen as an evolutionary marker in these large RNA genomes lying at the genome size boundary. A positive correlation exists between (+)RNA virus genome sizes and the presence of the RNA helicase and the ExoN domains. Although the mechanistic basis of the presence of the helicase is still unclear, the role of the ExoN activity has been linked to the existence of an RNA synthesis proofreading system. In large Nidovirales, ExoN is bound to a processive replicative RNA-dependent RNA polymerase (RdRp) and corrects mismatched bases during viral RNA synthesis. Over the last decade, a view of the overall process has been refined in Coronaviruses, and in particular in our lab (Ferron et al., PNAS, 2018). We have identified genetic markers of large RNA genomes that we wish to use to data-mine currently existing metagenomic datasets. We have also initiated a collaboration to sequence and explore new viromes that will be searched according to these criteria. Likewise, we have a collection of purified viral RdRps that are currently being used to generate RNA synthesis products that will be compared to existing NGS datasets of cognate viruses. We will be able to have an idea about how much genetic diversity is possibly achievable by viral RdRp (‘tunable fidelity’) versus the detectable diversity (i.e. after selection in the infected cell) that is actually produced.

Error-prone replication of a 5-formylcytosine-mediated DNA-peptide cross-link in human cells

DNA-protein cross-links can interfere with chromatin architecture, block DNA replication and transcription, and interfere with DNA repair. Here we synthesized a DNA 23-mer containing a site-specific DNA-peptide cross-link (DpC) by cross-linking an 11-mer peptide to the DNA epigenetic mark 5-formylcytosine in synthetic DNA and used it to generate a DpC-containing plasmid construct. Upon replication of the DpC-containing plasmid in HEK 293T cells, approximately 9% of progeny plasmids contained targeted mutations and 5% semitargeted mutations. Targeted mutations included C→T transitions and C deletions, whereas semitargeted mutations included several base substitutions and deletions near the DpC lesion. To identify DNA polymerases involved in DpC bypass, we comparatively studied translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with TLS polymerase knockouts or knockdowns. Knockdown of either hPol ι or hPol ζ reduced the mutation frequency by nearly 50%. However, the most significant reduction in mutation frequency (50%-70%) was observed upon simultaneous knockout of hPol η and hPol κ with knockdown of hPol ζ, suggesting that these TLS polymerases play a critical role in error-prone DpC bypass. Because TLS efficiency of the DpC construct was not significantly affected in TLS polymerase-deficient cells, we examined a possible role of replicative DNA polymerases in their bypass and determined that hPol δ and hPol ϵ can accurately bypass the DpC. We conclude that both replicative and TLS polymerases can bypass this DpC lesion in human cells but that mutations are induced mainly by TLS polymerases.

Protection of abasic sites during DNA replication by a stable thiazolidine protein-DNA cross-link.

Abasic (AP) sites are one of the most common DNA lesions that block replicative polymerases. HMCES recognizes and processes these lesions in the context of single-stranded DNA (ssDNA). A HMCES DNA-protein crosslink (DPC) intermediate is thought to shield the AP site from endonucleases and error-prone polymerases. The highly evolutionarily conserved SRAP domain of HMCES and its Escherichia coli ortholog YedK mediate lesion recognition. Here we discover the basis of AP site protection by SRAP domains from a crystal structure of the YedK DPC. YedK forms a stable thiazolidine linkage between a ring-opened AP site and the α-amino and sulfhydryl substituents of its N-terminal cysteine residue. The thiazolidine linkage explains the remarkable stability of the HMCES DPC, its resistance to strand cleavage, and the proteolysis requirement for resolution. Furthermore, its structure reveals that HMCES has specificity for AP sites in ssDNA at junctions found when replicative polymerases encounter the AP lesion.

Nucleoside Analogs with Selective Antiviral Activity against Dengue Fever and Japanese Encephalitis Viruses.

Dengue virus (DENV) and Japanese encephalitis virus (JEV) are important arthropod-borne viruses from the Flaviviridae family. DENV is a global public health problem with significant social and economic impacts, especially in tropical and subtropical areas. JEV is a neurotropic arbovirus endemic to east and southeast Asia. There are no U.S. FDA-approved antiviral drugs available to treat or to prevent DENV and JEV infections, leaving nearly one-third of the world's population at risk for infection. Therefore, it is crucial to discover potent antiviral agents against these viruses. Nucleoside analogs, as a class, are widely used for the treatment of viral infections. In this study, we discovered nucleoside analogs that possess potent and selective anti-JEV and anti-DENV activities across all serotypes in cell-based assay systems. Both viruses were susceptible to sugar-substituted 2'-C-methyl analogs with either cytosine or 7-deaza-7-fluoro-adenine nucleobases. Mouse studies confirmed the anti-DENV activity of these nucleoside analogs. Molecular models were assembled for DENV serotype 2 (DENV-2) and JEV RNA-dependent RNA polymerase replication complexes bound to nucleotide inhibitors. These models show similarities between JEV and DENV-2, which recognize the same nucleotide inhibitors. Collectively, our findings provide promising compounds and a structural rationale for the development of direct-acting antiviral agents with dual activity against JEV and DENV infections.

The presence of ribonucleotides in DNA has an ambiguous impact on the maintenance of genetic stability

High replication fidelity, understood as the DNA polymerases’ ability to select nucleotides with both correct base and sugar, is of critical importance for maintaining the genetic stability. Due to the fact that the cellular levels of ribonucleotides are much higher than the concentrations of deoxyribonucleotides, replicative polymerases are able to incorporate ribonucleotides with up to 1000-fold higher frequency than mismatched deoxyribonucleotides. The ability to discriminate against ribonucleotides by the DNA polymerases relies on the steric gate residue in the enzyme’s catalytic centre. Despite the fact that ribonucleotides are the most abundantly inserted incorrect nucleotides in DNA, they are not observed in properly functioning cells. The major pathway responsible for the recognition and removal of ribonucleotides from DNA is called Ribonucleotide Excision Repair. The impairment of ribonucleotide removal pathways can cause increased mutation rate, replication stress, DNA breakage, problems with transcription, chromatin structure maintenance, genetic disorders and cell death. In spite of that, ribonucleotide incorporation into DNA may have some positive biological impact, stimulating mismatch repair and non-homologous end joining.

Biochemical reconstitution of UV-induced mutational processes.

We reconstituted two biochemical processes that may contribute to UV-induced mutagenesis in vitro and analysed the mutational profiles in the products. One process is translesion synthesis (TLS) by DNA polymerases (Pol) δ, η and ζ, which creates C>T transitions at pyrimidine dimers by incorporating two dAMPs opposite of the dimers. The other process involves spontaneous deamination of cytosine, producing uracil in pyrimidine dimers, followed by monomerization of the dimers by secondary UV irradiation, and DNA synthesis by Pol δ. The mutational spectrum resulting from deamination without translesion synthesis is similar to a mutational signature found in melanomas, suggesting that cytosine deamination encountered by the replicative polymerase has a prominent role in melanoma development. However, CC>TT dinucleotide substitution, which is also commonly observed in melanomas, was produced almost exclusively by TLS. We propose that both TLS-dependent and deamination-dependent mutational processes are likely involved in UV-induced melanoma development.

DNA Rereplication Is Susceptible to Nucleotide-Level Mutagenesis.

The sources of genome instability, a hallmark of cancer, remain incompletely understood. One potential source is DNA rereplication, which arises when the mechanisms that prevent the reinitiation of replication origins within a single cell cycle are compromised. Using the budding yeast Saccharomyces cerevisiae, we previously showed that DNA rereplication is extremely potent at inducing gross chromosomal alterations and that this arises in part because of the susceptibility of rereplication forks to break. Here, we examine the ability of DNA rereplication to induce nucleotide-level mutations. During normal replication these mutations are restricted by three overlapping error-avoidance mechanisms: the nucleotide selectivity of replicative polymerases, their proofreading activity, and mismatch repair. Using lys2InsEA14 , a frameshift reporter that is poorly proofread, we show that rereplication induces up to a 30× higher rate of frameshift mutations and that this mutagenesis is due to passage of the rereplication fork, not secondary to rereplication fork breakage. Rereplication can also induce comparable rates of frameshift and base-substitution mutations in a more general mutagenesis reporter CAN1, when the proofreading activity of DNA polymerase ε is inactivated. Finally, we show that the rereplication-induced mutagenesis of both lys2InsEA14 and CAN1 disappears in the absence of mismatch repair. These results suggest that mismatch repair is attenuated during rereplication, although at most sequences DNA polymerase proofreading provides enough error correction to mitigate the mutagenic consequences. Thus, rereplication can facilitate nucleotide-level mutagenesis in addition to inducing gross chromosomal alterations, broadening its potential role in genome instability.

Origin of mutations in genes associated with human glioblastoma multiform cancer: random polymerase errors versus deamination

The etiology of glioblastoma multiforme (GBM), the most serious form of brain cancer, remains obscure, although it has been proposed that cancer risk is a function of random polymerase errors that occur during stem cell division and the resulting mutations in oncogenes and tumor suppressor genes. Analysis of the 8 genes (PTEN, TP53, EGFR, PIK3R1, PIK3CA, NF1, RB1, IDH1) that are mutated in at least 5% of GBM tumors indicates a non-random mutation pattern that reflects a significant role for hydrolytic deamination at CpG sites. The formation of activating mutations in some genes, e.g., IDH1, where a very limited set of mutations are oncogenic, statistically cannot involve random mutagenesis due to polymerase errors that occur during each stem cell replication. Comparison of the in vitro misincorporation tendencies of three replicative polymerases and the “random” mutation pattern in a subset of genes indicates non-polymerase based pathways are involved. Analysis of the mutation patterns shows that chemical deamination that occurs at a slow rate at each CpG is favored over random polymerase errors by a factor of more than 10 million. Therefore, if a truncating nonsense mutation in a tumor suppressor, or an activating missense mutation in an oncogene, can occur due to a C > T base substitution at a CpG sequence, it is highly favored over other mutation pathways.

High Sensitivity of Human Translesion DNA Synthesis Polymerase κ to Variation in O6-Carboxymethylguanine Structures

Carboxymethylation of DNA, including the formation of the DNA adduct O6-carboxymethylguanine ( O6-CMG), is associated with lifestyle factors, such as diet. It can impede replicative polymerases (Pols) and lead to replication fork stalling, or an alternative means for replication to proceed by translesion DNA synthesis (TLS). TLS requires specialized DNA Pols characterized by open and preformed active sites capable of preferential bypass of alkylated DNA adducts but that have high error rates, leading to mutations. Human TLS Pols can bypass O6-CMG with varying degrees of accuracy, but it is not known how the chemical structure of the O6-CMG adduct influences polymerase proficiency or fidelity. To better understand how adduct structure determines dNTP selection at lesion sites, we prepared DNA templates with a series of O6-CMG structural analogs and compared the primer extension patterns of Y- and X-family Pols in response to these modifications. The results indicate that the structure of the DNA adduct had a striking effect on dNTP selection by Pol κ and that an increased steric size influences the fidelity of Pol η, whereas Pol ι and β function were only marginally affected. To test the hypothesis that specific hydrogen bonding interactions between the templating base and the incoming dNTP are a basis of this selection, we modeled the structural analogs with incoming dNTP in the Pol κ active site. These data indicate that the base pairing geometry and stabilization by a dense hydrogen bonding network are important molecular features for dNTP incorporation, providing a basis for understanding error-free bypass of O6-CMG by Pol κ.

The Rev1-Polζ translesion synthesis mutasome: Structure, interactions and inhibition.

DNA contains information that must be safeguarded, but also accessed for transcription and replication. To perform replication, eukaryotic cells use the B-family DNA polymerase enzymes Polδ and Polɛ, which are optimized for accuracy, speed, and processivity. The molecular basis of these high-performance characteristics causes these replicative polymerases to fail at sites of DNA damage (lesions), which would lead to genomic instability and cell death. To avoid this, cells possess additional DNA polymerases such as the Y-family of polymerases and the B-family member Polζ that can replicate over sites of DNA damage in a process called translesion synthesis (TLS). While able to replicate over DNA lesions, the TLS polymerases exhibit low-fidelity on undamaged DNA and, consequently, must be prevented from replicating DNA under normal circumstances and recruited only when necessary. The replicative bypass of most types of DNA lesions requires the consecutive action of these specialized TLS polymerases assembled into a dynamic multiprotein complex called the Rev1/Polζ mutasome. To this end, posttranslational modifications and a network of protein-protein interactions mediated by accessory domains/subunits of the TLS polymerases control the assembly and rearrangements of the Rev1/Polζ mutasome and recruitment of TLS proteins to sites of DNA damage. This chapter focuses on the structures and interactions that control these processes underlying the function of the Rev1/Polζ mutasome, as well as the development of small molecule inhibitors of the Rev1/Polζ-dependent TLS holding promise as a potential anticancer therapy.

Mechanistic insights into the enzymatic activity and inhibition of the replicative polymerase exonuclease domain from Mycobacterium tuberculosis

DNA replication fidelity maintains low mutation rates in bacteria. The ε-subunit of a replisome generally acts as the main proofreader during replication, using its 3′–5′ exonuclease activity to excise misincorporated bases thereby maintaining faithful replication. In Mycobacterium tuberculosis (Mtb), however, the polymerase and histidinol phosphatase (PHP) domain of the DNA polymerase DnaE1 is the primary proofreader. This domain thus maintains low mutation rates during replication and is an attractive target for drug development. Even though the structures of DnaE polymerases are available from various organisms, including Mtb, the mechanism of exonuclease activity remains elusive. In this study, we sought to unravel the mechanism and also to identify scaffolds that can specifically inhibit the exonuclease activity. To gain insight into the mode of action, we also characterized the PHP domain of the Mtb error-prone polymerase DnaE2 which shares a nearly identical active site with DnaE1-PHP. Kinetic and mutational studies allowed us to identify the critical residue involved in catalysis. Combined inhibition and computational studies also revealed a specific mode of inhibition of DnaE1-PHP by nucleoside diphosphates. Thus, this study lays the foundation for the rational design of novel inhibitors which target the Mtb replicative proofreader.

Telomeres Increasingly Develop Aberrant Structures in Aging Humans.

Telomeres progressively shorten with age, and it has been proposed that critically short and dysfunctional telomeres contribute to aging and aging-associated diseases in humans. For many years it was thought that telomere erosion was strictly a consequence of the "end replication problem," or the inability of replicative polymerases to completely duplicate linear DNA ends. It is becoming increasingly evident, however, that telomere shortening of cultured human cells is also caused because of other replication defects in telomeric repeats, those that cause fragile telomeres and other aberrant telomeric structures that can be detected on metaphase chromosomes. Whether these replication defects contribute to telomere erosion also in human tissues is currently unknown. By analyzing peripheral blood mononuclear cells from a total of 35 healthy subjects ranging in age from 23 to 101 years, we demonstrated that telomeres increasingly display aberrant structures with advancing donor age. Although the percentages of fragile telomeres increased only until adulthood, the percentages of chromosomes displaying sister telomere loss and sister telomere chromatid fusions increased consistently throughout the entire human life span. Our data, therefore, suggest that telomeric replication defects other than the end replication problem contribute to aging-associated telomere erosion in humans.