Block DNA Replication Research Articles

Synthesis and polymerase bypass studies of DNA-peptide and DNA-protein conjugates.

DNA-peptide (DpCs) and DNA-protein cross-links (DPCs) are DNA lesions formed when polypeptides and nuclear proteins become covalently trapped on DNA strands. DNA-protein cross-links are of enormous size and hence pose challenges to cell survival by blocking DNA replication, transcription, and repair. However, DPCs can undergo proteolytic degradation via various pathways to give shorter polypeptide chains (DpCs). The resulting DpC lesions are efficiently bypassed by translesion synthesis (TLS) DNA polymerases like κ, η, δ, etc., although polymerase bypass efficiency as well as correct base insertion depends heavily on size, sequence context, and position of peptides in DpCs. This chapter explores various synthetic methods to generate these lesions including detailed experimental procedures for the construction of DpCs and DPCs via reductive amination and oxime ligation. Further we describe biochemical experiments to investigate the effects of these lesions on DNA polymerase activity and fidelity.

Depletion of the mini-chromosome maintenance complex binding protein allows the progression of cytokinesis despite abnormal karyokinesis during the asexual development of Plasmodium falciparum.

The eukaryotic cell cycle is typically divided into distinct phases with cytokinesis immediately following mitosis. To ensure proper cell division, each phase is tightly coordinated via feedback controls named checkpoints. During its asexual replication cycle, the malaria parasite Plasmodium falciparum undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division with intact nuclear envelope. The multi-nucleated schizont is then subjected to a single round of cytokinesis that produces dozens of daughter cells called merozoites. To date, no cell cycle checkpoints have been identified that regulate the Plasmodium spp. mode of division. Here, we identify the Plasmodium homologue of the Mini-Chromosome Maintenance Complex Binding Protein (PfMCMBP), which co-purified with the Mini-Chromosome Maintenance (MCM) complex, a replicative helicase required for genomic DNA replication. By conditionally depleting PfMCMBP, we disrupt nuclear morphology and parasite proliferation without causing a block in DNA replication. By immunofluorescence microscopy, we show that PfMCMBP depletion promotes the formation of mitotic spindle microtubules with extensions to more than one DNA focus and abnormal centrin distribution. Strikingly, PfMCMBP-deficient parasites complete cytokinesis and form aneuploid merozoites with variable cellular and nuclear sizes. Our study demonstrates that the parasite lacks a robust checkpoint response to prevent cytokinesis following aberrant karyokinesis.

Abstract PO-130: The mitochondrial protein ATAD3A promotes cisplatin resistance in oral squamous cell carcinoma

Abstract Cisplatin is well-known for its damaging effect on nuclear DNA, leading to cell death due to blockage of DNA replication and transcription, but this activity cannot fully explain why cisplatin displays cytotoxicity in post-mitotic tissues, suggesting other mechanisms may be in play. Many studies have challenged the current model of cisplatin resistance, however, the precise mechanism behind is still unknown. Here, we report that the mitochondrial protein ATAD3A functions as an oncogene in oral squamous cell carcinoma (OSCC), promoting cell proliferation and enhancing the ability of colony formation and anchorage-independent growth. Moreover, ATAD3A is involved in cisplatin resistance in OSCC, and knockdown of ATAD3A enhanced the sensitivity of OSCC cells to cisplatin. It appears that ATAD3A-mediated cisplatin resistance is regulated by other mechanisms rather than its expression levels, as evidenced by overexpression of a Walker A (WA) dead mutant of ATAD3A leads to a suppression in OSCC cell growth and resensitizes cisplatin-resistant OSCC cells to this drug, indicating a potent dominant-negative effect of defective ATP-binding of ATAD3A in chemotherapy. Our study demonstrates for the first time that ATAD3A plays an oncogenic role in OSCC and abrogating its ATP-binding ability may represent a promising regimen for better treatment of patients with OSCC, especially for those who suffer from cisplatin-resistance. Citation Format: Liwei Lang, Leslie Duncan, Caleb Jensen, Chloe Shay, Yong Teng. The mitochondrial protein ATAD3A promotes cisplatin resistance in oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-130.

The Relationship Between Actin Cytoskeleton and Membrane Transporters in Cisplatin Resistance of Cancer Cells

Cisplatin [cis-diamminedichloroplatinum (II)] is a platinum-based anticancer drug widely used for the treatment of various cancers. It forms interstrand and intrastrand cross-linking with DNA and block DNA replication, resulting in apoptosis. On the other hand, intrinsic and acquired cisplatin resistance restricts its therapeutic effects. Although some studies suggest that dramatic epigenetic alternations are involved in the resistance triggered by cisplatin, the mechanism is complicated and remains poorly understood. Recent studies reported that cytoskeletal structures regulate cisplatin sensitivity and that activities of membrane transporters contribute to the development of resistance to cisplatin. Therefore, we focus on the roles of actin filaments and membrane transporters in cisplatin-induced apoptosis. In this review, we summarize the relationship between actin cytoskeleton and membrane transporters in the cisplatin resistance of cancer cells.

Vascular endothelial cells mediated the indirect effect of mPEG-PCL copolymers on blood coagulation

Amphiphilic methoxy polyethyleneglycol-polycaprolactone (mPEG-PCL) block copolymer is widely used in the biomedical field for drug delivery or tissue engineering. At all events, mPEG-PCL copolymers in vivo applications would enter the blood circulation inevitably and contact with blood and vascular endothelial cells. However, previous studies on the hemocompatibility of biomedical polymers have only focused on the direct impact on blood components, ignoring the indirect impact on coagulation function mediated by the interaction between polymers and vascular endothelial cells. Hence, the influence of two mPEG-PCL copolymers (mPEG2k-PCL2k and mPEG5k-PCL2k) with different hydrophilic/hydrophobic ratio on human umbilical vein endothelial cells (HUVECs) and their mediated indirect impact on blood coagulation function were studied. The result showed that both mPEG-PCL copolymers displayed HUVECs proliferation suppression and increased NO release in a concentration-dependent manner. It was further confirmed by cellular uptake, cell cycle, and apoptosis measurement and found that mPEG-PCL copolymers could be endocytosed by HUVECs and significantly blocked DNA replication in G1 phase, but didn't cause obvious apoptosis. In comparison, mPEG2k-PCL2k had more impact on HUVECs than mPEG5k-PCL2k, attributing to the higher endocytosis efficiency of mPEG2k-PCL2k with higher hydrophobicity. Moreover, RT-PCR results of coagulation regulators and TEG measurements in human blood confirmed that mPEG-PCL induced HUVECs to downregulate the expression of anticoagulant factors and upregulate the expression of coagulation factors, thus accelerating blood coagulation and predicting thrombotic risk. This study provides new insights into the blood compatibility of polymeric carriers from the perspective of the vascular endothelium-mediated indirect effect of polymer on blood coagulation function.

Replisome bypass of transcription complexes and R-loops.

The vast majority of the genome is transcribed by RNA polymerases. G+C-rich regions of the chromosomes and negative superhelicity can promote the invasion of the DNA by RNA to form R-loops, which have been shown to block DNA replication and promote genome instability. However, it is unclear whether the R-loops themselves are sufficient to cause this instability or if additional factors are required. We have investigated replisome collisions with transcription complexes and R-loops using a reconstituted bacterial DNA replication system. RNA polymerase transcription complexes co-directionally oriented with the replication fork were transient blockages, whereas those oriented head-on were severe, stable blockages. On the other hand, replisomes easily bypassed R-loops on either template strand. Replication encounters with R-loops on the leading-strand template (co-directional) resulted in gaps in the nascent leading strand, whereas lagging-strand template R-loops (head-on) had little impact on replication fork progression. We conclude that whereas R-loops alone can act as transient replication blocks, most genome-destabilizing replication fork stalling likely occurs because of proteins bound to the R-loops.

Structural insight into FANCI-FANCD2 monoubiquitination.

The Fanconi anemia (FA) pathway coordinates a faithful repair mechanism for DNA damage that blocks DNA replication, such as interstrand cross-links. A key step in the FA pathway is the conjugation of ubiquitin on to FANCD2 and FANCI, which is facilitated by a large E3 ubiquitin ligase complex called the FA core complex. Mutations in FANCD2, FANCI or FA core complex components cause the FA bone marrow failure syndrome. Despite the importance of these proteins to DNA repair and human disease, our molecular understanding of the FA pathway has been limited due to a deficit in structural studies. With the recent development in cryo-electron microscopy (EM), significant advances have been made in structural characterization of these proteins in the last 6 months. These structures, combined with new biochemical studies, now provide a more detailed understanding of how FANCD2 and FANCI are monoubiquitinated and how DNA repair may occur. In this review, we summarize these recent advances in the structural and molecular understanding of these key components in the FA pathway, compare the activation steps of FANCD2 and FANCI monoubiquitination and suggest molecular steps that are likely to be involved in regulating its activity.

The ZGRF1 Helicase Promotes Recombinational Repair of Replication-Blocking DNA Damage in Human Cells

SUMMARYReplication-blocking DNA lesions are particularly toxic to proliferating cells because they can lead to chromosome mis-segregation if not repaired prior to mitosis. In this study, we report that ZGRF1 null cells accumulate chromosome aberrations following replication perturbation and show sensitivity to two potent replication-blocking anticancer drugs: mitomycin C and camptothecin. Moreover, ZGRF1 null cells are defective in catalyzing DNA damage-induced sister chromatid exchange despite accumulating excessive FANCD2, RAD51, and γ-H2AX foci upon induction of interstrand DNA crosslinks. Consistent with a direct role in promoting recombinational DNA repair, we show that ZGRF1 is a 5′-to-3′ helicase that catalyzes D-loop dissociation and Holliday junction branch migration. Moreover, ZGRF1 physically interacts with RAD51 and stimulates strand exchange catalyzed by RAD51-RAD54. On the basis of these data, we propose that ZGRF1 promotes repair of replication-blocking DNA lesions through stimulation of homologous recombination.

Interaction with single-stranded DNA-binding protein localizes ribonuclease HI to DNA replication forks and facilitates R-loop removal.

DNA replication complexes (replisomes) routinely encounter proteins and unusual nucleic acid structures that can impede their progress. Barriers can include transcription complexes and R-loops that form when RNA hybridizes with complementary DNA templates behind RNA polymerases. Cells encode several RNA polymerase and R-loop clearance mechanisms to limit replisome exposure to these potential obstructions. One such mechanism is hydrolysis of R-loops by ribonuclease HI (RNase HI). Here, we examine the cellular role of the interaction between Escherichia coli RNase HI and the single-stranded DNA-binding protein (SSB) in this process. Interaction with SSB localizes RNase HI foci to DNA replication sites. Mutation of rnhA to encode an RNase HI variant that cannot interact with SSB but that maintains enzymatic activity (rnhAK60E) eliminates RNase HI foci. The mutation also produces a media-dependent slow-growth phenotype and an activated DNA damage response in cells lacking Rep helicase, which is an enzyme that disrupts stalled transcription complexes. RNA polymerase variants that are thought to increase or decrease R-loop accumulation enhance or suppress, respectively, the growth phenotype of rnhAK60E rep::kan strains. These results identify a cellular role for the RNase HI/SSB interaction in helping to clear R-loops that block DNA replication.

The FANC/BRCA Pathway Releases Replication Blockades by Eliminating DNA Interstrand Cross-Links

DNA interstrand cross-links (ICLs) represent a major barrier blocking DNA replication fork progression. ICL accumulation results in growth arrest and cell death—particularly in cell populations undergoing high replicative activity, such as cancer and leukemic cells. For this reason, agents able to induce DNA ICLs are widely used as chemotherapeutic drugs. However, ICLs are also generated in cells as byproducts of normal metabolic activities. Therefore, every cell must be capable of rescuing lCL-stalled replication forks while maintaining the genetic stability of the daughter cells in order to survive, replicate DNA and segregate chromosomes at mitosis. Inactivation of the Fanconi anemia/breast cancer-associated (FANC/BRCA) pathway by inherited mutations leads to Fanconi anemia (FA), a rare developmental, cancer-predisposing and chromosome-fragility syndrome. FANC/BRCA is the key hub for a complex and wide network of proteins that—upon rescuing ICL-stalled DNA replication forks—allows cell survival. Understanding how cells cope with ICLs is mandatory to ameliorate ICL-based anticancer therapies and provide the molecular basis to prevent or bypass cancer drug resistance. Here, we review our state-of-the-art understanding of the mechanisms involved in ICL resolution during DNA synthesis, with a major focus on how the FANC/BRCA pathway ensures DNA strand opening and prevents genomic instability.

Inhibition of ubiquitin-specific protease 7 sensitizes acute myeloid leukemia to chemotherapy.

Resistance of acute myeloid leukemia (AML) to therapeutic agents is frequent. Consequently, the mechanisms leading to this resistance must be understood and addressed. In this paper, we demonstrate that inhibition of deubiquitinylase USP7 significantly reduces cell proliferation in vitro and in vivo, blocks DNA replication progression and increases cell death in AML. Transcriptomic dataset analyses reveal that a USP7 gene signature is highly enriched in cells from AML patients at relapse, as well as in residual blasts from patient-derived xenograft (PDX) models treated with clinically relevant doses of cytarabine, which indicates a relationship between USP7 expression and resistance to therapy. Accordingly, single-cell analysis of AML patient samples at relapse versus at diagnosis showed that a gene signature of the pre-existing subpopulation responsible for relapse is enriched in transcriptomes of patients with a high USP7 level. Furthermore, we found that USP7 interacts and modulates CHK1 protein levels and functions in AML. Finally, we demonstrated that USP7 inhibition acts in synergy with cytarabine to kill AML cell lines and primary cells of patients with high USP7 levels. Altogether, these data demonstrate that USP7 is both a marker of resistance to chemotherapy and a potential therapeutic target in overcoming resistance to treatment.

The roles of polymerases ν and θ in replicative bypass of O6- and N2-alkyl-2′-deoxyguanosine lesions in human cells

Exogenous and endogenous chemicals can react with DNA to produce DNA lesions that may block DNA replication. Not much is known about the roles of polymerase (Pol) ν and Pol θ in translesion synthesis (TLS) in cells. Here we examined the functions of these two polymerases in bypassing major-groove O6-alkyl-2'-deoxyguanosine (O6-alkyl-dG) and minor-groove N2-alkyl-dG lesions in human cells, where the alkyl groups are ethyl, n-butyl (nBu), and, for O6-alkyl-dG, pyridyloxobutyl. We found that Pol ν and Pol θ promote TLS across major-groove O6-alkyl-dG lesions. O6-alkyl-dG lesions mainly induced G→A mutations that were modulated by the two TLS polymerases and the structures of the alkyl groups. Simultaneous ablation of Pol ν and Pol θ resulted in diminished mutation frequencies for all three O6-alkyl-dG lesions. Depletion of Pol ν alone reduced mutations only for O6-nBu-dG, and sole loss of Pol θ attenuated the mutation rates for O6-nBu-dG and O6-pyridyloxobutyl-dG. Replication across the two N2-alkyl-dG lesions was error-free, and Pol ν and Pol θ were dispensable for their replicative bypass. Together, our results provide critical knowledge about the involvement of Pol ν and Pol θ in bypassing alkylated guanine lesions in human cells.

A coexistence of pemphigus vulgaris and type II diabetes in geriatric patient: a case report

Background: Pemphigus Vulgaris (PV) is an autoimmune bullous disorder that usually affected the middle-aged and elderly. PV management in elderly patients is still a challenge for clinicians because of an increased risk of developing diabetes in the elderly. Corticosteroid is the first-line treatment for PV; however, careful consideration should be taken for diabetes patients due to the risk of disrupting glucose control leading to acute decompensation.Case: A seventy-year-old female with type II diabetes reported to Sanglah General Hospital with multiple painful blisters on the chest, back, and oral mucous. She has been treated with insulin since last year, but the treatment was non-compliant. Dermatological examination showed multiple flaccid bullae containing serous fluid with a positive Nikolsky sign, and multiple skin erosions. Histopathology finding is appropriate with PV. The Tzank smear examination revealed acantholytic cells. She was diagnosed with PV and typed II diabetes; therefore, we treated her with azathioprine, insulin and Lantus injection; sodium chloride 0.9% compress and fusidic acid cream 2% twice daily for topical treatment.Conclusion: Azathioprine is the treatment of choice because it works by blocking DNA replication without increasing blood glucose levels. The selection of appropriate medications can improve patient prognosis.

Cross-linking of the DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of cisplatin

1,1,2,2-cis-diamminedichloroplatinum (II) (cisplatin) is a chemotherapeutic agent widely used in the clinic to treat various cancers. The antitumor activity of cisplatin is generally attributed to its ability to form intrastrand and interstrand DNA-DNA cross-links via sequential platination of two nucleophilic sites within the DNA duplex. However, cisplatin also induces DNA- protein lesions (DPCs) that may contribute to its biological effects due to their ability to block DNA replication and transcription. We previously reported that over 250 nuclear proteins including high mobility group proteins, histone proteins, and elongation factors formed DPCs in human HT1080 cells treated with cisplatin (Ming et al. Chem. Res. Toxicol. 2017, 30, 980–995). Interestingly, cisplatin-induced DNA-protein conjugates were reversed upon heating, by an unknown mechanism. In the present work, DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) was used as a model to investigate the molecular details of cisplatin-mediated DNA-protein cross-linking and to establish the mechanism of their reversal. We found that AGT is readily cross-linked to DNA in the presence of cisplatin. HPLC-ESI+-MS/MS sequencing of tryptic peptides originating from dG-Pt-AGT complexes revealed that the cross-linking occurred at six sites within this protein including Glu110, Lys125, Cys145, His146, Arg147, and Cys150. Cisplatin-induced Lys-Gua cross-links (1,1-cis-diammine-2-(5-amino-5-carboxypentyl)amino-2-(2'-deoxyguanosine-7-yl)-platinum(II) (dG-Pt-Lys) were detected by HPLC-ESI+-MS/MS of total digests of modified protein in comparison with the corresponding authentic standard. Upon heating, dG-Pt-AGT complexes were subject to platination migration from protein to DNA, forming cis-[Pt(NH3)2{d(GpG)}] cross-links which were detected by HPLC-ESI+-MS/MS. Our results provide a new insight into the mechanism of cisplatin-mediated DNA-protein cross-linking and their dynamic equilibrium with the corresponding DNA-DNA lesions.

Kinetics of DNA Adducts and Abasic Site Formation in Tissues of Mice Treated with a Nitrogen Mustard.

Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the alkylation of DNA bases. NM-adducts block DNA replication in cancer cells by forming cytotoxic DNA interstrand cross-links. We previously characterized several adducts formed by reaction of bis(2-chloroethyl)ethylamine (NM) with calf thymus (CT) DNA and the MDA-MB-231 mammary tumor cell line. The monoalkylated N7-guanine (NM-G) adduct and its cross-link (G-NM-G) were major lesions. The cationic NM-G undergoes a secondary reaction through depurination to form an apurinic (AP) site or reacts with hydroxide to yield the stable ring-opened N5-substituted formamidopyrimidine (NM-Fapy-G) adduct. Both of these lesions are mutagenic and may contribute to secondary tumor development, a major clinical limitation of NM chemotherapy. We established a kinetic model with NM-treated female mice and measured the rates of formation and removal of NM-DNA adducts and AP sites. We employed liquid chromatography-mass spectrometry (LC-MS) to measure NM-G, G-NM-G, and NM-Fapy-G adducts in liver, lung, and spleen over 168 h. NM-G reached a maximum level within 6 h in all organs and then rapidly declined. The G-NM-G cross-link and NM-FapyG were more persistent with half-lives over three-times longer than NM-G. We quantified AP site lesions in the liver and showed that NM treatment increased AP site levels by 3.7-fold over the basal levels at 6 h. The kinetics of AP site repair closely followed the rate of removal of NM-G; however, AP sites remained 1.3-fold above basal levels 168 h post-treatment with NM. Our data provide new insights into NM-induced DNA damage and biological processing in vivo. The quantitative measurement of the spectrum of NM adducts and AP sites can serve as biomarkers in the design and assessment of the efficacy of novel chemotherapeutic regimens.

FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair.

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2–FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryoEM to determine the structure of recombinant chicken FANCD2 and FANCI complexes. FANCD2–FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA. Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2–FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.

Regulation of translesion DNA synthesis in mammalian cells.

The genomes of all living cells are under endogenous and exogenous attacks every day, causing diverse genomic lesions. Most of the lesions can be timely repaired by multiple DNA repair pathways. However, some may persist during S-phase, block DNA replication, and challenge genome integrity. Eukaryotic cells have evolved DNA damage tolerance (DDT) to mitigate the lethal effects of arrested DNA replication without prior removal of the offending DNA damage. As one important mode of DDT, translesion DNA synthesis (TLS) utilizes multiple low-fidelity DNA polymerases to incorporate nucleotides opposite DNA lesions to maintain genome integrity. Three different mechanisms have been proposed to regulate the polymerase switching between high-fidelity DNA polymerases in the replicative machinery and one or more specialized enzymes. Additionally, it is known that proliferating cell nuclear antigen (PCNA) mono-ubiquitination is essential for optimal TLS. Given its error-prone property, TLS is closely associated with spontaneous and drug-induced mutations in cells, which can potentially lead to tumorigenesis and chemotherapy resistance. Therefore, TLS process must be tightly modulated to avoid unwanted mutagenesis. In this review, we will focus on polymerase switching and PCNA mono-ubiquitination, the two key events in TLS pathway in mammalian cells, and summarize current understandings of regulation of TLS process at the levels of protein-protein interactions, post-translational modifications as well as transcription and noncoding RNAs. Environ. Mol. Mutagen. 61:680-692, 2020. © 2020 Wiley Periodicals, Inc.

Bioreducible and traceable Ru(III) prodrug-loaded mesoporous silica nanoparticles for sequentially targeted nonsmall cell lung cancer chemotherapy

Ruthenium-based agents are promising in cancer chemotherapy but lack specific targeting and controlled release properties. Herein, a new class of bioreducible mer-[Ru(III)Cl3(dmso)(L)] prodrugs with highly DNA binding affinity were designed and synthesized for nucleus-targeted chemotherapy and selective activation in response to the tumor microenvironment. In this regard, ruthenium(III) prodrugs were reduced into highly active ruthenium(II) drugs in response to high intracellular glutathione levels, resulting in tumor apoptosis through synergistic intercalation that blocked DNA replication and coordination that cleaved nuclear DNA. In light of this, a multifunctional nanodrug (Ru-MSN-PLip) was constructed by integrating RuDPNI prodrug-loaded mesoporous silica nanoparticles (Ru-MSN) with a fusion protein-incorporated liposome (PLip) to achieve multiple targeting and eGFP-based fluorescence imaging of nonsmall cell lung cancer. Due to fusion protein-based cancer cell recognition and Ru(III) prodrug-mediated nuclear DNA targeting, Ru-MSN-PLip achieved sequential delivery and greater antitumor activity. In addition, Ru-MSN-PLip exhibited long-term blood circulation and selective accumulation in tumors, leading to significantly limited tumor growth in vivo without notable toxicity. Our findings indicated the potential applications of these types of bioreducible and traceable nanoprodrugs for efficient and safe nonsmall cell lung cancer therapy.

Escherichia coli YcaQ is a DNA glycosylase that unhooks DNA interstrand crosslinks.

Interstrand DNA crosslinks (ICLs) are a toxic form of DNA damage that block DNA replication and transcription by tethering the opposing strands of DNA. ICL repair requires unhooking of the tethered strands by either nuclease incision of the DNA backbone or glycosylase cleavage of the crosslinked nucleotide. In bacteria, glycosylase-mediated ICL unhooking was described in Streptomyces as a means of self-resistance to the genotoxic natural product azinomycin B. The mechanistic details and general utility of glycosylase-mediated ICL repair in other bacteria are unknown. Here, we identify the uncharacterized Escherichia coli protein YcaQ as an ICL repair glycosylase that protects cells against the toxicity of crosslinking agents. YcaQ unhooks both sides of symmetric and asymmetric ICLs in vitro, and loss or overexpression of ycaQ sensitizes E. coli to the nitrogen mustard mechlorethamine. Comparison of YcaQ and UvrA-mediated ICL resistance mechanisms establishes base excision as an alternate ICL repair pathway in bacteria.

Cooler temperatures slow the repair of DNA damage in tadpoles exposed to ultraviolet radiation: Implications for amphibian declines at high altitude.

Ultraviolet B radiation (UVBR) damages the DNA of exposed cells, causing dimers to form between adjacent pyrimidine nucleotides. These dimers block DNA replication, causing mutations and apoptosis. Most organisms utilize biochemical or biophysical DNA repair strategies to restore DNA structure; however, as with most biological reactions, these processes are likely to be thermally sensitive. Tadpoles exposed to elevated UVBR at low environmental temperatures have significantly higher rates of mortality and developmental deformities compared with tadpoles exposed to the same levels of UVBR at higher environmental temperatures. We hypothesized that low environmental temperatures impair the primary enzymatic (photolyase) DNA repair pathway in amphibians, leading to the accumulation of DNA damage. To test this hypothesis, we compared DNA repair rates and photolyase gene expression patterns in Limnodynastes peronii. Tadpoles were acutely exposed to UVBR for 1hr at either 20 or 30°C, and we measured DNA damage and photolyase expression levels at intervals following this exposure. Temperature had a significant effect on the rate of DNA repair, with repair at 30°C occurring twice as fast as repair at 20°C. Photolyase gene expression (6-4 PP and CPD) was significantly upregulated by UVBR exposure, with expression levels increasing within 6hr of UVBR exposure. CPD expression levels were not significantly affected by temperature, but 6-4 PP expression was significantly higher in tadpoles in the 30°C treatment within 12hr of UVBR exposure. These data support the hypothesis that DNA repair rates are thermally sensitive in tadpoles and may explain why enigmatic amphibian declines are higher in montane regions where UVBR levels are naturally elevated and environmental temperatures are lower.