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Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad175'?/5'? embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad175'?/5'? ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents. [ABSTRACT FROM AUTHOR]

Cellular and humoral defence mechanisms are essential for the survival of individuals and species. Thus, DNA repair prevents mutations and cytotoxicity from DNA damage, thereby reducing the risks of inappropriate cell death, developmental defects, premature ageing and cancer. Similarly, antigen-dependent acquired immune responses prevent infections and also have a role in cancer prevention. DNA repair is highly complex and functions in an intricate network that also involves transcription, replication, cell cycle regulation, and the immune system. DNA damage is repaired by at least four major mechanisms, each requiring many different proteins. In addition there are “subpathways”, and back-up mechanisms both within and between pathways. Various defects in DNA repair result in different forms of cancer, e.g. the rare syndrome Xeroderma pigmentosum and the more common diseases early-onset breast cancer and hereditary non-polyposis colon cancer. Surprisingly, recent research has revealed molecular interactions between the ancient DNA repair mechanisms and the much younger acquired immune system. Thus, the classical base excision enzyme uracil-DNA glycosylase encoded by the UNG gene is also involved in somatic hypermutation and class switch recombination, e.g. from IgM antibodies to IgG, yielding secreted high affinity antibodies. Mutations in both alleles of UNG result in a hyper-IgM syndrome with life-threatening infections. Furthermore, it has recently become clear that not only DNA, but also RNA and proteins are repaired. Thus, certain aberrant methylations in RNA are repaired by oxidative demethylation in one step restoring the normal base, and at least in a bacterial model system this increases survival several-fold after exposure to methylating agents. Proteins are repaired both at the peptide amino acid level and at the structural level. RNA and protein repair are likely to be important to prevent the formation of cytotoxic protein aggregates of the types known to cause neurodegenerative diseases e.g. Alzheimer’s, Parkinson’s and Huntington’s diseases, and other diseases as well. In conclusion, recent research has demonstrated an unexpected complexity of cellular defence mechanisms that function in intricate networks, rather than as independent mechanisms. The new knowledge opens for interventions that are based on a deeper understanding of the mechanisms of defence. [ABSTRACT FROM AUTHOR]

Eucaryotic DNA replication complex is now one of the most intensively studied subjects of molecular biology and biochemistry In addition to detailed studies on the structures and functions of individual DNA polymerases involved in this process, other enzymes and protein factors are also given much attention. The structures and functions of proteins in the replication complexes are studied by various approaches, including X-ray diffraction analysis. At present, this approach provides sufficient information about the structures and functions of individual biopolymers and their complexes with ligands. However, this approach is unsuitable for studies on proteins, which cannot be cloned and isolated in amounts sufficient for X-ray diffraction analysis. Moreover, this approach is inapplicable for studies on multicomponent systems, such as DNA replication and repair complexes. Furthermore, data of X-ray diffraction analysis virtually never characterize the variety of dynamic interactions in enzymatic systems. Affinity modification is an alternative and rather successful approach for studies on structure-functional organization of supramolecular structures. This approach can be used for studies on individual enzymes and their complexes with substrates and also on systems consisting of numerous interacting proteins and nucleic acids. The purpose of this review is to analyze the available data obtained by affinity modification studies on the eucaryotic replication complex. [ABSTRACT FROM AUTHOR]

A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2′-position of the ribose fragment of the 5′-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure. [ABSTRACT FROM AUTHOR]

DNA polymerase ℇ (Pol ℇ) of Saccharomyces cerevisiae participates in many aspects of DNA replication, as well as in DNA repair. In order to clarify molecular mechanisms employed in the multiple tasks of Pol ℇ, we have been characterizing the interaction between Pol ℇ and DNA. Analysis of the four-subunit Pol ℇ complex by gel mobility shift assay revealed that the complex binds not only to single-stranded (ss) DNA but also equally well to double-stranded (ds) DNA. A truncated polypeptide consisting of the N-terminal domain of Pol2p catalytic subunit binds to ssDNA but not to dsDNA, indicating that the Pol2p C-terminal domain and/or the auxiliary subunits are involved in the dsDNA-binding. The dsDNA-binding by Pol ℇ does not require DNA ends or specific DNA sequences. Further analysis by competition experiments indicated that Pol ℇ contains at least two distinct DNA-binding sites, one of which binds exclusively to ssDNA and the other to dsDNA. The dsDNA-binding site, however, is suggested to also bind ssDNA. The DNA polymerase activity of Pol ℇ is inhibited by ssDNA but not by dsDNA. Furthermore, purification of the Pol ℇ auxiliary subunits Dpb3p and Dpb4p revealed that these proteins form a heterodimer and associate with dsDNA. Pol ℇ has multiple sites at which it interacts with DNA. One of these sites has a strong affinity for dsDNA, a feature that is not generally associated with DNA polymerases. Involvement of the Dpb3p-Dpb4p complex in the dsDNA-binding of Pol ℇ is inferred. [ABSTRACT FROM AUTHOR]

Replication of eukaryotic DNA is performed by a protein complex in which the central part is played by DNA polymerases. Synthesis with eukaryotic DNA polymerases α, δ, and ℇ involves various replication factors, including the replication protein A, replication factor C, proliferating cell nuclear antigen, etc. Replication enzymes and factors also participate in DNA repair, which is interrelated with DNA replication. The function of the entire multicomponent system is regulated by protein–nucleic acid and protein–protein interactions. The eukaryotic replication complex was not isolated as a stable supramolecular structure, suggesting its dynamic organization. Hence X-ray analysis and other instrumental techniques are hardly suitable for studying this system. An alternative approach is affinity modification. Its most promising version involves in situ generation of photoreactive DNA replication intermediates. The review considers the recent progress in photoaffinity modification studies of DNA polymerases, eukaryotic replication factors, and their interactions with DNA replication intermediates. [ABSTRACT FROM AUTHOR]

The pol3-13 mutation is located in the C-terminal end of POL3, the gene encoding the catalytic subunit of polymerase δ, and confers thermosensitivity onto the Saccharomyces cerevisiae mutant strain. To get insight about DNA replication control, we performed a genetic screen to identify genes that are synthetic lethal with pol3-13. Mutations in genes encoding the two other subunits of DNA polymerase δ (HYS2, POL32) were identified. Mutations in two recombination genes (RAD50, RAD51) were also identified, confirming that homologous recombination is necessary for pol3-13 mutant strain survival. Other mutations were identified in genes involved in repair and genome stability (MET18/MMS19), in the control of origin-firing and/or transcription (ABF1, SRB7), in the S/G2 checkpoint (RAD53), in the Ras-cAMP signal transduction pathway (MKS1), in nuclear pore metabolism (SEH1), in protein degradation (DOC1) and in folding (YDJ1). Finally, mutations in three genes of unknown function were isolated (NBP35, DRE2, TAH18). Synthetic lethality between pol3-13 and each of the three mutants pol32, mms19 and doc1 could be suppressed by a rad18 deletion, suggesting an important role of ubiquitination in DNA replication control. We propose that the pol3-13 mutant generates replicative problems that need both homologous recombination and an intact checkpoint machinery to be overcome. [ABSTRACT FROM AUTHOR]

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway. [ABSTRACT FROM AUTHOR]

A number of intrinsic and extrinsic mutagens induce structural damage in cellular DNA. These DNA damages are cytotoxic, miscoding or both and are believed to be at the origin of cell lethality, tissue degeneration, ageing and cancer. In order to counteract immediately the deleterious effects of such lesions, leading to genomic instability, cells have evolved a number of DNA repair mechanisms including the direct reversal of the lesion, sanitation of the dNTPs pools, mismatch repair and several DNA excision pathways including the base excision repair (BER) nucleotide excision repair (NER) and the nucleotide incision repair (NIR). These repair pathways are universally present in living cells and extremely well conserved. This review is focused on the repair of lesions induced by free radicals and ionising radiation. The BER pathway removes most of these DNA lesions, although recently it was shown that other pathways would also be efficient in the removal of oxidised bases. In the BER pathway the process is initiated by a DNA glycosylase excising the modified and mismatched base by hydrolysis of the glycosidic bond between the base and the deoxyribose of the DNA, generating a free base and an abasic site (APsite) which in turn is repaired since it is cytotoxic and mutagenic. [ABSTRACT FROM AUTHOR]

Over the past few years, several new 3′?5′ exonucleases have been identified. In vitro studies of these enzymes have uncovered much about their potential functions in vivo, and certain organisms with a defect in 3′?5′ exonucleases have an increased susceptibility to cancer, especially under conditions of stress. Here, we look at not only the newly discovered enzymes, but also at the roles of other 3′?5′ exonucleases in the quality control of DNA synthesis, where they act as proofreading exonucleases for DNA polymerases during DNA replication, repair and recombination. [ABSTRACT FROM AUTHOR]

The 8-oxo-7,8-dihydroguanine, referred to as 8-oxoG, is a highly mutagenic DNA lesion that can provoke the appearance of mismatches if it escapes the DNA Damage Response. The specific recognition of its structural signature by the hOGG1 glycosylase is the first step along the Base Excision Repair pathway, which ensures the integrity of the genome by preventing the emergence of mutations. 8-oxoG formation, structural features, and repair have been matters of extensive research; more recently, this active field of research expended to the more complicated case of 8-oxoG within clustered lesions. Indeed, the presence of a second lesion within 1 or 2 helix turns can dramatically impact the repair yields of 8-oxoG by glycosylases. In this work, we use μ s-range molecular dynamics simulations and machine-learning-based postanalysis to explore the molecular mechanisms associated with the recognition of 8-oxoG by hOGG1 when embedded in a multiple-lesion site with a mismatch in 5 ′ or 3 ′ . We delineate the stiffening of the DNA–protein interactions upon the presence of the mismatches, and rationalize the much lower repair yields reported with a 5 ′ mismatch by describing the perturbation of 8-oxoG structural features upon addition of an adjacent lesion. [ABSTRACT FROM AUTHOR]

During each cell duplication, the entirety of the genomic DNA in every cell must be accurately and quickly copied. Given the short time available for the chore, the requirement of many proteins, and the daunting amount of DNA present, DNA replication poses a serious challenge to the cell. A high level of coordination between polymerases and other DNA and chromatin-interacting proteins is vital to complete this task. One of the most important proteins for maintaining such coordination is PCNA. PCNA is a multitasking protein that forms a homotrimeric ring that encircles the DNA. It serves as a processivity factor for DNA polymerases and acts as a landing platform for different proteins interacting with DNA and chromatin. Therefore, PCNA is a signaling hub that influences the rate and accuracy of DNA replication, regulates DNA damage repair, controls chromatin formation during the replication, and the proper segregation of the sister chromatids. With so many essential roles, PCNA recruitment and turnover on the chromatin is of utmost importance. Three different, conserved protein complexes are in charge of loading/unloading PCNA onto DNA. Replication factor C (RFC) is the canonical complex in charge of loading PCNA during the S-phase. The Ctf18 and Elg1 (ATAD5 in mammalian) proteins form complexes similar to RFC, with particular functions in the cell's nucleus. Here we summarize our current knowledge about the roles of these important factors in yeast and mammals. [ABSTRACT FROM AUTHOR]

DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work performed in cell-free media, we studied the ability of selected DNA polymerases to overcome a monofunctional adduct of the cytotoxic/antitumor platinum–acridinylthiourea conjugate [PtCl(en)(L)](NO3)2 (en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) (ACR) in its favored 5′-CG sequence. We focused on how a single site-specific ACR adduct with intercalation potency affects the processivity and fidelity of DNA-dependent DNA polymerases involved in translesion synthesis (TLS) and repair. The ability of the G(N7) hybrid ACR adduct formed in the 5′-TCGT sequence of a 24-mer DNA template to inhibit the synthesis of a complementary DNA strand by the exonuclease-deficient Klenow fragment of DNA polymerase I (KFexo−) and human polymerases eta, kappa, and iota was supplemented by thermodynamic analysis of the polymerization process. Thermodynamic parameters of a simulated translesion synthesis across the ACR adduct were obtained by using microscale thermophoresis (MST). Our results show a strong inhibitory effect of an ACR adduct on enzymatic TLS: there was only small synthesis of a full-length product (less than 10%) except polymerase eta (~20%). Polymerase eta was able to most efficiently bypass the ACR hybrid adduct. Incorporation of a correct dCMP opposite the modified G residue is preferred by all the four polymerases tested. On the other hand, the frequency of misinsertions increased. The relative efficiency of misinsertions is higher than that of matched cytidine monophosphate but still lower than for the nonmodified control duplex. Thermodynamic inspection of the simulated TLS revealed a significant stabilization of successively extended primer/template duplexes containing an ACR adduct. Moreover, no significant decrease of dissociation enthalpy change behind the position of the modification can contribute to the enzymatic TLS observed with the DNA-dependent, repair-involved polymerases. This TLS could lead to a higher tolerance of cancer cells to the ACR conjugate compared to its enhanced analog, where thiourea is replaced by an amidine group: [PtCl(en)(L)](NO3)2 (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine). [ABSTRACT FROM AUTHOR]

The POLQ gene encodes DNA polymerase θ, a 2590 amino acid protein product harboring DNA-dependent ATPase, template-dependent DNA polymerase, dNTP-dependent endonuclease, and 5′–dRP lyase functions. Polymerase θ participates at an essential step of a DNA double-strand break repair pathway able to join 5′-resected substrates by locating and pairing microhomologies present in 3′-overhanging single-stranded tails, cleaving the extraneous 3′-DNA by dNTP-dependent end-processing, before extending the nascent 3′ end from the microhomology annealing site. Metazoans require polymerase θ for full resistance to DNA double-strand break inducing agents but can survive knockout of the POLQ gene. Cancer cells with compromised homologous recombination, or other DNA repair defects, over-utilize end-joining by polymerase θ and often over-express the POLQ gene. This dependency points to polymerase θ as an ideal drug target candidate and multiple drug-development programs are now preparing to enter clinical trials with small-molecule inhibitors. Specific inhibitors of polymerase θ would not only be predicted to treat BRCA-mutant cancers, but could thwart accumulated resistance to current standard-of-care cancer therapies and overcome PARP-inhibitor resistance in patients. This article will discuss synthetic lethal strategies targeting polymerase θ in DNA damage-response-deficient cancers and summarize data, describing molecular structures and enzymatic functions. [ABSTRACT FROM AUTHOR]

Burkholderia gladioli has high versatility and adaptability to various ecological niches. Here, we constructed a pan-genome using 14 genome sequences of B. gladioli, which originate from different niches, including gladiolus, rice, humans, and nature. Functional roles of core and niche-associated genomes were investigated by pathway enrichment analyses. Consequently, we inferred the uniquely important role of niche-associated genomes in (1) selenium availability during competition with gladiolus host; (2) aromatic compound degradation in seed-borne and crude oil-accumulated environments, and (3) stress-induced DNA repair system/recombination in the cystic fibrosis-niche. We also identified the conservation of the rhizomide biosynthetic gene cluster in all the B. gladioli strains and the concentrated distribution of this cluster in human isolates. It was confirmed the absence of complete CRISPR/Cas system in both plant and human pathogenic B. gladioli and the presence of the system in B. gladioli living in nature, possibly reflecting the inverse relationship between CRISPR/Cas system and virulence. [ABSTRACT FROM AUTHOR]

Xeroderma pigmentosum variant (XP-V) represents one of the most common forms of this cancer-prone DNA repair syndrome. Unlike classical XP cells, XP-V cells are normal in nucleotide excision repair but defective in post-replication repair. The precise molecular defect in XP-V is currently unknown, but it appears to be a protein involved in translesion synthesis. Here we established a sensitive assay system using an SV40 origin-based plasmid to detect XP-V complementation activity. Using this system, we isolated a protein from HeLa cells capable of complementing the defects in XP-V cell extracts. The protein displays novel DNA polymerase activity which replicates cyclobutane pyrimidine dimer-containing DNA templates. The XPV polymerase activity was dependent on MgCl2, sensitive to NEM, moderately sensitive to KCl, resistant to both aphidicolin and ddTTP, and not stimulated by PCNA. In glycerol density gradients, the activity co-sedimented with a 54 kDa polypeptide at 3.5S, indicating that the monomeric form of this poly-peptide was responsible for the activity. The protein factor corrected the translesion defects of extracts from three XPV cell strains. Bypass DNA synthesis by the XP-V polymerase occurred only in the presence of dATP, indicating that it can incorporate only dATP to bypass a di-thymine lesion. [ABSTRACT FROM AUTHOR]

Examines the types of DNA adducts formed by the major classes of chemotherapeutic agents. Repair of genotoxic lesions; Role of repair in the resistance to chemotherapeutic agents; Clinical strategies for modulating repair activity.

The DNA polymerase accessory factor proliferating cell nuclear antigen (PCNA) has been caught in interaction with an ever increasing number of proteins. To characterize the sites and functions of some of these interactions, we constructed four mutants of human PCNA and analysed them in a variety of assays. By targeting loops on the surface of the PCNA trimer and changing three or four residues at a time to alanine, we found that a region including part of the domain-connecting loop of PCNA and loops on one face of the trimer, close to the C-termini, is involved in binding to all of the following proteins: DNA polymerase δ, replication factor C, the flap endonuclease Fen1, the cyclin dependent kinase inhibitor p21 and DNA ligase I. An inhibition of DNA ligation caused by the interaction of PCNA with DNA ligase I was found, and we show that DNA ligase I and Fen1 can inhibit DNA synthesis by DNA polymerase δ/PCNA. We demonstrate that PCNA must be located below a 5′ flap on a forked template to stimulate Fen1 activity, and considering the interacting region on PCNA for Fen1, this suggests an orientation for PCNA during DNA replication with the C-termini facing forwards, in the direction of DNA synthesis. [ABSTRACT FROM AUTHOR]

Cisplatin is a widely used antineoplastic agent, whose efficacy is limited by primary and acquired therapeutic resistance. Recently, a bladder cancer genome-wide CRISPR/Cas9 knock-out screen correlated cisplatin sensitivity to multiple genetic biomarkers. Among the screen's top hits was the HECT domain-containing ubiquitin E3 ligase (HUWE1). In this review, HUWE1 is postulated as a therapeutic response modulator, affecting the collision between platinum-DNA adducts and the replication fork, the primary cytotoxic action of platins. HUWE1 can alter the cytotoxic response to platins by targeting essential components of the DNA damage response including BRCA1, p53, and Mcl-1. Deficiency of HUWE1 could lead to enhanced DNA damage repair and a dysfunctional apoptotic apparatus, thereby inducing resistance to platins. Future research on the relationship between HUWE1 and platins could generate new mechanistic insights into therapy resistance. Ultimately, HUWE1 might serve as a clinical biomarker to tailor cancer treatment strategies, thereby improving cancer care and patient outcomes. [ABSTRACT FROM AUTHOR]

While Next-Generation Sequencing (NGS) and technological advances have been useful in identifying genetic profiles of tumorigenesis, novel target proteins and various clinical biomarkers, cancer continues to be a major global health threat. DNA replication, DNA damage response (DDR) and repair, and cell cycle regulation continue to be essential systems in targeted cancer therapies. Although many genes involved in DDR are known to be tumor suppressor genes, cancer cells are often dependent and addicted to these genes, making them excellent therapeutic targets. In this review, genes implicated in DNA replication, DDR, DNA repair, cell cycle regulation are discussed with reference to peptide or small molecule inhibitors which may prove therapeutic in cancer patients. Additionally, the potential of utilizing novel synthetic lethal genes in these pathways is examined, providing possible new targets for future therapeutics. Specifically, we evaluate the potential of TONSL as a novel gene for targeted therapy. Although it is a scaffold protein with no known enzymatic activity, the strategy used for developing PCNA inhibitors can also be utilized to target TONSL. This review summarizes current knowledge on non-oncogene addiction, and the utilization of synthetic lethality for developing novel inhibitors targeting non-oncogenic addiction for cancer therapy. [ABSTRACT FROM AUTHOR]

In their life cycle, plants are exposed to various unfavorable environmental factors including ultraviolet (UV) radiation emitted by the Sun. UV-A and UV-B, which are partially absorbed by the ozone layer, reach the surface of the Earth causing harmful effects among the others on plant genetic material. The energy of UV light is sufficient to induce mutations in DNA. Some examples of DNA damage induced by UV are pyrimidine dimers, oxidized nucleotides as well as single and double-strand breaks. When exposed to light, plants can repair major UV-induced DNA lesions, i.e., pyrimidine dimers using photoreactivation. However, this highly efficient light-dependent DNA repair system is ineffective in dim light or at night. Moreover, it is helpless when it comes to the repair of DNA lesions other than pyrimidine dimers. In this review, we have focused on how plants cope with deleterious DNA damage that cannot be repaired by photoreactivation. The current understanding of light-independent mechanisms, classified as dark DNA repair, indispensable for the maintenance of plant genetic material integrity has been presented. [ABSTRACT FROM AUTHOR]

The eukaryotic genome is in a constant state of modification and repair. Faithful transmission of the genomic information from parent to daughter cells depends upon an extensive system of surveillance, signaling, and DNA repair, as well as accurate synthesis of DNA during replication. Often, replicative synthesis occurs over regions of DNA that have not yet been repaired, presenting further challenges to genomic stability. DNA polymerase δ (pol δ) occupies a central role in all of these processes: catalyzing the accurate replication of a majority of the genome, participating in several DNA repair synthetic pathways, and contributing structurally to the accurate bypass of problematic lesions during translesion synthesis. The concerted actions of pol δ on the lagging strand, pol ϵ on the leading strand, associated replicative factors, and the mismatch repair (MMR) proteins results in a mutation rate of less than one misincorporation per genome per replication cycle. This low mutation rate provides a high level of protection against genetic defects during development and may prevent the initiation of malignancies in somatic cells. This review explores the role of pol δ in replication fidelity and genome maintenance. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The majority of DNA polymerases (DNAPs) are specialized enzymes with specific roles in DNA replication, translesion DNA synthesis (TLS), or DNA repair. The enzymatic characteristics to perform accurate DNA replication are in apparent contradiction with TLS or DNA repair abilities. For instance, replicative DNAPs incorporate nucleotides with high fidelity and processivity, whereas TLS DNAPs are low-fidelity polymerases with distributive nucleotide incorporation. Plant organelles (mitochondria and chloroplast) are replicated by family-A DNA polymerases that are both replicative and TLS DNAPs. Furthermore, plant organellar DNA polymerases from the plant model Arabidopsis thaliana (AtPOLIs) execute repair of double-stranded breaks by microhomology-mediated end-joining and perform Base Excision Repair (BER) using lyase and strand-displacement activities. AtPOLIs harbor three unique insertions in their polymerization domain that are associated with TLS, microhomology-mediated end-joining (MMEJ), strand-displacement, and lyase activities. We postulate that AtPOLIs are able to execute those different functions through the acquisition of these novel amino acid insertions, making them multifunctional enzymes able to participate in DNA replication and DNA repair. [ABSTRACT FROM AUTHOR]

Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication and repair. It forms a homotrimeric ring that embraces the DNA and slides along it, anchoring DNA polymerases and other DNA editing enzymes. It also interacts with regulatory proteins through a sequence motif known as PCNA Interacting Protein box (PIP-box). We here review the latest contributions to knowledge regarding the structure-function relationships in human PCNA, particularly the mechanism of sliding, and of the molecular recognition of canonical and non-canonical PIP motifs. The unique binding mode of the oncogene p15 is described in detail, and the implications of the recently discovered structure of PCNA bound to polymerase δ are discussed. The study of the post-translational modifications of PCNA and its partners may yield therapeutic opportunities in cancer treatment, in addition to illuminating the way PCNA coordinates the dynamic exchange of its many partners in DNA replication and repair. [ABSTRACT FROM AUTHOR]

Since the discovery of the DNA double helix, there has been a fascination in understanding the molecular mechanisms and cellular processes that account for: (i) the transmission of genetic information from one generation to the next and (ii) the remarkable stability of the genome. Nucleic acid biologists have endeavored to unravel the mysteries of DNA not only to understand the processes of DNA replication, repair, recombination, and transcription but to also characterize the underlying basis of genetic diseases characterized by chromosomal instability. Perhaps unexpectedly at first, DNA helicases have arisen as a key class of enzymes to study in this latter capacity. From the first discovery of ATP-dependent DNA unwinding enzymes in the mid 1970's to the burgeoning of helicase-dependent pathways found to be prevalent in all kingdoms of life, the story of scientific discovery in helicase research is rich and informative. Over four decades after their discovery, we take this opportunity to provide a history of DNA helicases. No doubt, many chapters are left to be written. Nonetheless, at this juncture we are privileged to share our perspective on the DNA helicase field – where it has been, its current state, and where it is headed. [ABSTRACT FROM AUTHOR]

Deleterious mutations in Breast Cancer 1 (BRCA1) are associated with an increased risk of breast and ovarian cancer. Mutations in the tandem BRCA1 C-terminal (tBRCT) protein domain disrupt critical protein interactions required for the faithful repair of DNA through homologous recombination, which contributes to oncogenesis. Our studies have identified RICTOR, PRR5, and SIN1 subunits of the mammalian target of rapamycin complex 2 (mTORC2) as interacting partners with the tBRCT domain of BRCA1 leading to the disruption of the mTORC2 complex. However, the interplay between mTORC2 signaling and BRCA1 function in the DNA damage response (DDR) remains to be determined. In this study, we used protein interaction assays to determine the binary interactions between the tBRCT domain and mTORC2 subunits, evaluated the impact of mTOR inhibition on the transcriptional function of the tBRCT, evaluated the impact of mTOR signaling on BRCA1 recruitment to DNA damage-induced foci and determined the breast cancer cell line response to mTOR inhibition dependent upon BRCA1 expression and mutation. This study determined that PRR5, RICTOR, and SIN1 could each independently interact with the BRCA1 tBRCT. Inhibition of mTORC1, but not mTORC1/2, increases BRCA1 transcriptional activation activity. Treatment with pan-mTOR inhibitor PP242 diminishes DNA damage-induced γH2AX and BRCA1 foci formation. Breast cancer cells lacking expression of functional BRCA1 are more sensitive to mTOR inhibitors. These data suggest that mTOR signaling is required for BRCA1 response to DNA damage and breast cancer cells lacking BRCA1 are more sensitive to pan-mTOR inhibition. This work suggests chemotherapeutic strategies using mTOR inhibitors could be tailored for patients that lack functional BRCA1. [ABSTRACT FROM AUTHOR]

Maintenance of genome integrity is a key process in all organisms. DNA polymerases (Pols) are central players in this process as they are in charge of the faithful reproduction of the genetic information, as well as of DNA repair. Interestingly, all eukaryotes possess a large repertoire of polymerases. Three protein complexes, DNA Pol α, δ, and ε, are in charge of nuclear DNA replication. These enzymes have the fidelity and processivity required to replicate long DNA sequences, but DNA lesions can block their progression. Consequently, eukaryotic genomes also encode a variable number of specialized polymerases (between five and 16 depending on the organism) that are involved in the replication of damaged DNA, DNA repair, and organellar DNA replication. This diversity of enzymes likely stems from their ability to bypass specific types of lesions. In the past 10–15 years, our knowledge regarding plant DNA polymerases dramatically increased. In this review, we discuss these recent findings and compare acquired knowledge in plants to data obtained in other eukaryotes. We also discuss the emerging links between genome and epigenome replication. [ABSTRACT FROM AUTHOR]

Methylation of cytosine (5-meC) is a critical epigenetic modification in many eukaryotes, and genomic DNA methylation landscapes are dynamically regulated by opposed methylation and demethylation processes. Plants are unique in possessing a mechanism for active DNA demethylation involving DNA glycosylases that excise 5-meC and initiate its replacement with unmodified C through a base excision repair (BER) pathway. Plant BER-mediated DNA demethylation is a complex process involving numerous proteins, as well as additional regulatory factors that avoid accumulation of potentially harmful intermediates and coordinate demethylation and methylation to maintain balanced yet flexible DNA methylation patterns. Active DNA demethylation counteracts excessive methylation at transposable elements (TEs), mainly in euchromatic regions, and one of its major functions is to avoid methylation spreading to nearby genes. It is also involved in transcriptional activation of TEs and TE-derived sequences in companion cells of male and female gametophytes, which reinforces transposon silencing in gametes and also contributes to gene imprinting in the endosperm. Plant 5-meC DNA glycosylases are additionally involved in many other physiological processes, including seed development and germination, fruit ripening, and plant responses to a variety of biotic and abiotic environmental stimuli. [ABSTRACT FROM AUTHOR]

7,8-Dihydro-8-oxoguanine (oxoG) is the most abundant oxidative DNA lesion with dual coding properties. It forms both Watson–Crick (anti)oxoG:(anti)C and Hoogsteen (syn)oxoG:(anti)A base pairs without a significant distortion of a B-DNA helix. DNA polymerases bypass oxoG but the accuracy of nucleotide incorporation opposite the lesion varies depending on the polymerase-specific interactions with the templating oxoG and incoming nucleotides. High-fidelity replicative DNA polymerases read oxoG as a cognate base for A while treating oxoG:C as a mismatch. The mutagenic effects of oxoG in the cell are alleviated by specific systems for DNA repair and nucleotide pool sanitization, preventing mutagenesis from both direct DNA oxidation and oxodGMP incorporation. DNA translesion synthesis could provide an additional protective mechanism against oxoG mutagenesis in cells. Several human DNA polymerases of the X- and Y-families efficiently and accurately incorporate nucleotides opposite oxoG. In this review, we address the mutagenic potential of oxoG in cells and discuss the structural basis for oxoG bypass by different DNA polymerases and the mechanisms of the recognition of oxoG by DNA glycosylases and dNTP hydrolases. [ABSTRACT FROM AUTHOR]

Oxidative stress and the resulting damage to DNA are inevitable consequence of endogenous physiological processes further amplified by cellular responses to environmental exposures. If left unrepaired, oxidative DNA lesions can block essential processes such as transcription and replication or can induce mutations. Emerging data also indicate that oxidative base modifications such as 8-oxoG in gene promoters may serve as epigenetic marks, and/or provide a platform for coordination of the initial steps of DNA repair and the assembly of the transcriptional machinery to launch adequate gene expression alterations. Here, we briefly review the current understanding of oxidative lesions in genome stability maintenance and regulation of basal and inducible transcription. [ABSTRACT FROM AUTHOR]

Human lung cancer H1299 (p53-null) cells often display enhanced susceptibility to chemotherapeutics comparing to A549 (p53-wt) cells. However, little is known regarding to the association of DNA damage-response (DDR) pathway heterogeneity with drug sensitivity in these two cells. We investigated the DDR pathway differences between A549 and H1299 cells exposed to 8-chloro-adenosine (8-Cl-Ado), a potential anticancer drug that can induce DNA double-strand breaks (DSBs), and found that the hypersensitivity of H1299 cells to 8-Cl-Ado is associated with its DSB overaccumulation. The major causes of excessive DSBs in H1299 cells are as follows: First, defect of p53-p21 signal and phosphorylation of SMC1 increase S phase cells, where replication of DNA containing single-strand DNA break (SSB) produces more DSBs in H1299 cells. Second, p53 defect and no available induction of DNA repair protein p53R2 impair DNA repair activity in H1299 cells more severely than A549 cells. Third, cleavage of PARP-1 inhibits topoisomerase I and/or topoisomerase I-like activity of PARP-1, aggravates DNA DSBs and DNA repair mechanism impairment in H1299 cells. Together, DDR pathway heterogeneity of cancer cells is linked to cancer susceptibility to DNA damage-based chemotherapeutics, which may provide aid in design of chemotherapy strategy to improve treatment outcomes. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS), generated as a by-product of mitochondrial oxidative phosphorylation, are particularly damaging to the genome of skeletal muscle because of their high oxygen consumption. Proliferating myoblasts play a key role during muscle regeneration by undergoing myogenic differentiation to fuse and restore damaged muscle. This process is severely impaired during aging and in muscular dystrophies. In this study, we investigated the role of oxidatively damaged DNA and its repair in the mitochondrial genome of proliferating skeletal muscle progenitor myoblasts cells and their terminally differentiated product, myotubes. Using the C2C12 cell line as a well-established model for skeletal muscle differentiation, we show that myoblasts are highly sensitive to ROS-mediated DNA damage, particularly in the mitochondrial genome, due to deficiency in 5’ end processing at the DNA strand breaks. Ectopic expression of the mitochondrial-specific 5’ exonuclease, EXOG, a key DNA base excision/single strand break repair (BER/SSBR) enzyme, in myoblasts but not in myotubes, improves the cell’s resistance to oxidative challenge. We linked loss of myoblast viability by activation of apoptosis with deficiency in the repair of the mitochondrial genome. Moreover, the process of myoblast differentiation increases mitochondrial biogenesis and the level of total glutathione. We speculate that our data may provide a mechanistic explanation for depletion of proliferating muscle precursor cells during the development of sarcopenia, and skeletal muscle dystrophies. [ABSTRACT FROM AUTHOR]

Summary Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5′ end of the repair gap. When the 5′ end is blocked and not amenable to β-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4−/− mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species. [ABSTRACT FROM AUTHOR]