SciCombinator

Discover the most talked about and latest scientific content & concepts.

Journal: DNA repair

0

Topoisomerase I (Top1) removes DNA torsional stress by nicking and resealing one strand of DNA, and is essential in higher eukaryotes. The enzyme is frequently overproduced in tumors and is the sole target of the chemotherapeutic drug camptothecin (CPT) and its clinical derivatives. CPT stabilizes the covalent Top1-DNA cleavage intermediate, which leads to toxic double-strand breaks (DSBs) when encountered by a replication fork. In the current study, we examined genetic instability associated with CPT treatment or with Top1 overexpression in the yeast Saccharomyces cerevisiae. Two types of instability were monitored: Top1-dependent deletions in haploid strains, which do not require processing into a DSB, and instability at the repetitive ribosomal DNA (rDNA) locus in diploid strains, which reflects DSB formation. Three 2-bp deletion hotspots were examined and mutations at each were elevated either when a wild-type strain was treated with CPT or when TOP1 was overexpressed, with the mutation frequency correlating with the level of TOP1 overexpression. Under both conditions, deletions at novel positions were enriched. rDNA stability was examined by measuring loss-of-heterozygosity and as was observed previously upon CPT treatment of a wild-type strain, Top1 overexpression destabilized rDNA. We conclude that too much, as well as too little of Top1 is detrimental to eukaryotic genomes, and that CPT has destabilizing effects that extend beyond those associated with DSB formation.

Concepts: DNA, Gene, Genetics, Bacteria, Genome, Fungus, DNA replication, Saccharomyces cerevisiae

0

Translesion synthesis (TLS) is the mechanism in which DNA polymerases (TLS polymerases) bypass unrepaired template damage with high error rates. DNA polymerase η and ζ (Polη and Polζ) are major TLS polymerases that are conserved from yeast to humans. In this study, we quantified frequencies of base-substitutions by yeast Polη and Polζ on undamaged and abasic templates in vitro. For accurate quantification, we used a next generation sequencing (NGS)-based method where DNA products were directly analyzed by parallel sequencing. On undamaged templates, Polη and Polζ showed distinct base-substitution profiles, and the substitution frequencies were differently influenced by the template sequence. The base-substitution frequencies were influenced mainly by the adjacent bases both upstream and downstream of the substitution sites. Thus we present the base-substitution signatures of these polymerases in a three-base format. On templates containing abasic sites, Polη created deletions at the lesion in more than 50% of the TLS products, but the formation of the deletions was suppressed by the presence of Polζ. Polζ and Polη cooperatively facilitated the TLS reaction over an abasic site in vitro, suggesting that these two polymerases can cooperate in efficient and high fidelity TLS.

Concepts: DNA, DNA replication, Reverse transcriptase, DNA polymerase, DNA polymerase I, Polymerase, DNA polymerase III holoenzyme, EC 2.7.7

0

Only mammalian apurinic/apyrimidinic endonuclease1 (APE1) has been reported to possess both DNA repair and redox activities. C terminal of the protein is required for base excision repair, while the redox activity resides in the N terminal due to cysteine residues at specific positions. APE1s from other organisms studied so far lack the redox activity in spite of having the N terminal domain. We find that APE1 from the Cnidarian Hydra exhibits both endonuclease and redox activities similar to mammalian APE1. We further show the presence of the three indispensable cysteines in Hydra APE1 for redox activity by site directed mutagenesis. Importance of redox domain but not the repair domain of APE1 in regeneration has been demonstrated by using domain-specific inhibitors. Our findings clearly demonstrate that the redox function of APE1 evolved very early in metazoan evolution and is not a recent acquisition in mammalian APE1 as believed so far.

Concepts: DNA, Gene, Mutation, Bacteria, Evolution, Metabolism, Organism, DNA repair

0

imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli.

Concepts: DNA, Gene, Genetics, Bacteria, Evolution, Flagellum, Proteobacteria, Caulobacter crescentus

0

DNA nuclease/helicase 2 (DNA2), a multi-functional protein protecting the high fidelity of genomic transmission, plays critical roles in DNA replication and repair processes. In the maturation of Okazaki fragments, DNA2 acts synergistically with other enzymes to cleave the DNA-RNA primer flaps via different pathways. DNA2 is also involved in the stability of mitochondrial DNA and the maintenance of telomeres. Moreover, DNA2 potentially participates in controlling the cell cycle by repairing the DNA replication faults at main checkpoints. In addition, previous evidences demonstrated that DNA2 also functions in the repair process of DNA damages, such as base excision repair (BER). Currently, large studies revealed the structures and functions of DNA2 in prokaryotes and unicellular eukaryotes, such as bacteria and yeast. However, the studies that highlighted the functions of human DNA2 (hDNA2) and the relationships with other multifunctional proteins are still elusive, and more precise investigations are immensely needed. Therefore, this review mainly encompasses the key functions of DNA2 in human cells with various aspects, especially focusing on the genome integrity, and also generalizes the recent insights to the mechanisms related to the occurrence of cancer and other diseases potentially linked to the mutations in DNA2.

Concepts: DNA, Protein, Cell nucleus, Cell, Bacteria, Eukaryote, DNA repair, DNA replication

0

If unrepaired, damage to genomic DNA can cause mutations and/or be cytotoxic. Single base lesions are repaired via the base excision repair (BER) pathway. The first step in BER is the recognition and removal of the nucleobase lesion by a glycosylase enzyme. For example, human oxoguanine glycosylase 1 (hOGG1) is responsible for removal of the prototypic oxidatively damaged nucleobase, 8-oxo-7,8-dihydroguanine (8-oxoG). To date, most studies of glycosylases have used free duplex DNA substrates. However, cellular DNA is packaged as repeating nucleosome units, with 145 base pair segments of DNA wrapped around histone protein octamers. Previous studies revealed inhibition of hOGG1 at the nucleosome dyad axis and in the absence of chromatin remodelers. In this study, we reveal that even in the absence of chromatin remodelers or external cofactors, hOGG1 can initiate BER at positions off the dyad axis and that this activity is facilitated by spontaneous and transient unwrapping of DNA from the histones. Additionally, we find that solution accessibility as determined by hydroxyl radical footprinting is not fully predictive of glycosylase activity and that histone tails can suppress hOGG1 activity. We therefore suggest that local nuances in the nucleosome environment and histone-DNA interactions can impact glycosylase activity.

Concepts: DNA, Gene, Genetics, Histone, DNA repair, Nucleosome, Chromatin, Oxoguanine glycosylase

0

Although chromosome aberrations are known to derive from distance-dependent mis-rejoining of chromosome fragments, evaluating whether a certain model describes such “proximity effects” better than another one is complicated by the fact that different approaches have often been tested under different conditions. Herein, a biophysical model (“BIANCA”, i.e. BIophysical ANalysis of Cell death and chromosome Aberrations) was upgraded, implementing explicit chromosome-arm domains and two new models for the dependence of the rejoining probability on the fragment initial distance, r. Such probability was described either by an exponential function like exp(-r/r0), or by a Gaussian function like exp(-r(2)/2σ(2)), where r0 and σ were adjustable parameters. The second, and last, parameters was the yield of “Cluster Lesions” (CL), where “Cluster Lesion” defines a critical DNA damage producing two independent chromosome fragments. The model was applied to low-LET-irradiated lymphocytes (doses: 1-4Gy) and fibroblasts (1-6.1Gy). Good agreement with experimental yields of dicentrics and centric rings, and thus their ratio (“F-ratio”), was found by both the exponential model (with r0=0.8μm for lymphocytes and 0.7μm for fibroblasts) and the Gaussian model (with σ=1.1μm for lymphocytes and 1.3μm for fibroblasts). While the former also allowed reproducing dose-responses for excess acentric fragments, the latter substantially underestimated the experimental curves. Both models provided G-ratios (ratio of acentric to centric rings) higher than those expected from randomness, although the values calculated by the Gaussian model were lower than those calculated by the exponential one. For lymphocytes the calculated G-ratios were in good agreement with the experimental ones, whereas for fibroblasts both models substantially underestimated the experimental results, which deserves further investigation. This work suggested that, although both models performed better than a step model (which previously allowed reproducing the F-ratio but underestimated the G-ratio), an exponential function describes proximity effects better than a Gaussian one.

Concepts: Cytogenetics, Chromosomes, Real number, Normal distribution, Aneuploidy, Exponential growth, Exponentials, Proportionality

0

Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25bp dsDNA or 25bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35bp blunt ended dsDNA) or 25bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes.

Concepts: DNA, Protein, Gene, Transcription, DNA repair, Telomere, Non-homologous end joining, DNA-PKcs

0

O(6)-Methylguanine (O(6)-MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O(6)-MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O(6)-MeG by gp90 exo(-). O(6)-MeG partially inhibited full-length extension by gp90 exo(-). O(6)-MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo(-) extends beyond T:O(6)-MeG 2-fold more efficiently than C:O(6)-MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O(6)-MeG show fast burst phases. The pre-steady-state incorporation efficiency (kpol/Kd,dNTP) is decreased in the order of dCTP:G>dTTP:O(6)-MeG>dCTP:O(6)-MeG. The presence of O(6)-MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg(2+). Misincorporation of dTTP opposite O(6)-MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O(6)-MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O(6)-MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1.

Concepts: DNA, Mutation, Bacteria, Pseudomonas aeruginosa, Phage therapy, DNA replication, Pseudomonas, DNA polymerase

0

The presence of an enhancer element, RD(INK4/ARF) (RD), in the prominent INK4-ARF locus provides a novel en bloc mechanism to simultaneously regulate the transcription of the p15(INK4B) (p15), p16(INK4A) (p16), and p14(ARF) tumor suppressor genes. While genetic inactivation of p15, p16, and p14(ARF) in human cancers has been extensively studied, little is known about RD alteration and its potential contributions to cancer progression. In this review, we discuss recent developments in RD alteration and its association with p15, p16, and p14(ARF) alterations in human cancers, and demonstrate that RD deletion may represent a novel mechanism to simultaneously down-regulate p15, p16, and p14(ARF), thus promoting carcinogenesis.

Concepts: DNA, Gene, Genetics, Gene expression, Cancer, Oncology, P53, Tumor suppressor gene