Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase.

Mammalian polynucleotide kinase (mPNK) is a critical DNA repair enzyme whose 5'-kinase and 3'-phoshatase activities function with poorly understood but striking specificity to restore 5'-phosphate/3'-hydroxyl termini at sites of DNA damage. Here we integrated site-directed mutagenesis and small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of mPNK. The flexible attachment of the FHA domain to the catalytic segment, elucidated by SAXS, enables the interactions of mPNK with diverse DNA substrates and protein partners required for effective orchestration of DNA end repair. Point mutations surrounding the kinase active site identified two substrate recognition surfaces positioned to contact distinct regions on either side of the phosphorylated 5'-hydroxyl. DNA substrates bind across the kinase active site cleft to position the double-stranded portion upstream of the 5'-hydroxyl on one side, and the 3'-overhang on the opposite side. The bipartite DNA-binding surface of the mPNK kinase domain explains its preference for recessed 5'-termini, structures that would be encountered in the course of DNA strand break repair.

Inducible repair of thymine glycol detected by an ultrasensitive assay for DNA damage.

An ultrasensitive assay for measuring DNA base damage is described that couples immunochemical recognition with capillary electrophoresis and laser-induced fluorescence detection. The method provides a detection limit of 3 x 10(-21) moles, an improvement of four to five orders of magnitude over current methods. Induction and repair of thymine glycols were studied in irradiated A549 cells (a human lung carcinoma cell line). Exposure of these cells to a low dose of radiation (0.25 Gray) 4 hours before a clinically relevant dose (2 Gray) enhanced removal of thymine glycols after the higher dose. These data provide evidence for an inducible repair response for radiation-induced damage to DNA bases.

Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line.

The radiosensitive rodent mutant cell line xrs-5 is defective in DNA double-strand break repair and lacks the Ku component of the DNA-activated protein kinase, DNA-PK. Here radiosensitive human cell lines were analyzed for DNA-PK activity and for the presence of related proteins. The radiosensitive human malignant glioma M059J cell line was found to be defective in DNA double-strand break repair, but fails to express the p350 subunit of DNA-PK. These results suggest that DNA-PK kinase activity is involved in DNA double-strand break repair.

Role of apoptosis in low-dose hyper-radiosensitivity.

Little is known about the mode of cell killing associated with low-dose hyper-radiosensitivity, the radiation response that describes the enhanced sensitivity of cells to small doses of ionizing radiation. Using a technique that measures the activation of caspase 3, we have established a relationship between apoptosis detected 24 h after low-dose radiation exposure and low-dose hyper-radiosensitivity in four mammalian cell lines (T98G, U373, MR4 and 3.7 cells) and two normal human lymphoblastoid cell lines. The existence of low-dose hyper-radiosensitivity in clonogenic survival experiments was found to be associated with an elevated level of apoptosis after low-dose exposures, corroborating earlier observations (Enns et al., Mol. Cancer Res. 2, 557-566, 2004). We also show that enriching populations of MR4 and V79 cells with G(1)-phase cells, to minimize the numbers of G(2)-phase cells, abolished the enhanced low-dose apoptosis. These cell-cycle enrichment experiments strengthen the reported association between low-dose hyper-sensitivity and the radioresponse of G(2)-phase cells. These data are consistent with our current hypothesis to explain low-dose hyper-radiosensitivity, namely that the enhanced sensitivity of cells to low doses of ionizing radiation reflects the failure of ATM-dependent repair processes to fully arrest the progression of damaged G(2)-phase cells harboring unrepaired DNA breaks entering mitosis.

Factors influencing the removal of thymine glycol from DNA in gamma-irradiated human cells.

The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.

Dual action of tirapazamine in the induction of DNA strand breaks.

Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-dioxide, SR 4233) is the lead compound of a new class of hypoxic cell cytotoxins showing considerable antitumor activity. Hypoxic cytotoxicity of tirapazamine is believed to be mediated by free radical attack of its one-electron reduced metabolite on DNA, but little is known about the DNA lesions induced by the drug. Using the anoxic xanthine/xanthine oxidase system to effect one-electron reduction of tirapazamine under controlled conditions, we studied the action of the drug toward pUC18 and calf thymus DNA. Agarose gel electrophoresis indicated that tirapazamine causes substantially higher levels of single-strand breakage than double-stand breakage. The 5' DNA termini at the single-strand breaks were shown to be phosphorylated. Little, if any, base damage was observed when the damaged DNA was analyzed by a 32P-postlabeling assay. The major detectable lesion (comprising approximately 32% of the 3' ends of tirapazamine-induced single-strand breaks) was the phosphoglycolate moiety, which is caused by deoxyribose fragmentation. Since phosphoglycolate formation requires the addition of oxygen, we conclude that tirapazamine acts in a dual fashion to produce phosphoglycolates: (a) to generate a free radical in the deoxyribose ring (i.e., .C-4' and (b) then to donate an oxygen atom. The oxygen donation by tirapazamine was confirmed by anoxic irradiation of DNA in the presence of the unmetabolized drug. Increasing the concentration of the drug (up to 50 microM) led to a dramatic increase in the yield of phosphoglycolate.

Methylene blue-mediated photooxidation of 7,8-dihydro-8-oxo-2'-deoxyguanosine.

One well known product of the methylene blue-mediated photosensitization of 2'-deoxyguanosine (dG) in oxygen saturated aqueous solution is 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG). We observed that the rate of 8-oxodG photodecomposition by methylene blue-mediated photosensitization is approx. 3-times faster than for dG. The primary products of the methylene blue-mediated photosensitization of 8-oxodG are 2-amino-5-((2-deoxy-beta-D-erythro-pentofuranosyl)amino)-4H-imidazol-4-o ne (dIz), 2,2-diamino-4-((2-deoxy-beta-D-erythro-pentofuranosyl)amino)-5(2H)-oxazo lone (dZ), the 4R* and 4S* diastereoisomers of 4,8-dihydro-4-hydroxy-8-oxo-2'-deoxyguanosine (dO), and an as yet unidentified product with a molecular weight of 287 (dX). Except for the latter product, these compounds have all been identified following the methylene blue-mediated photooxidation of dG. Methylene blue-mediated photooxidation of 8-oxodG in D2O instead of H2O leads to a 4-fold increase in the rate of 8-oxodG photodecomposition while the addition of sodium azide retards the reaction, observations which imply that the reaction occurs via a type II (singlet oxygen mediated) mechanism. Like 8-oxodG, dIz and dZ are sensitive to hot piperidine and likely contribute to strand breaks observed in double stranded DNA exposed to methylene blue plus light followed by hot piperidine. Because 8-oxodG generates predominately G-->T transversions, the photooxidation of 8-oxodG to dIz, dO, and dX may explain the predominance of G-->C transversions in single-stranded M13mp2 bacteriophage DNA exposed to methylene blue plus light and then transfected into SOS-induced Escherichia coli.

Relative affinities of poly(ADP-ribose) polymerase and DNA-dependent protein kinase for DNA strand interruptions.

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are important nuclear enzymes that cooperate to minimize genomic damage caused by DNA strand interruptions. DNA strand interruptions trigger the ADP-ribosylation activity and phosphorylation activity of PARP and DNA-PK respectively. In order to understand the relationship of PARP and DNA-PK with respect to DNA binding required for their activation, we analyzed the kinetics of the reactions and determined the apparent dissociation constants (Kd app) of the enzymes for DNA strand interruptions. PARP has a high binding affinity for blunt ends of DNA (Kd app=116 pM) and 3' single-base overhangs (Kd app=332 pM) in comparison to long overhangs (Kd app=2.6-5.0 nM). Nicks are good activators of PARP although the affinity of PARP for nicks (Kd app=467 pM) is 4-fold less than that for blunt ends. The Kd app of DNA-PK for 3' single-base overhangs, blunt ends and long overhangs is 704 pM, 1.3 nM and 1.4-2.2 nM respectively. These results demonstrate that (1) PARP, when compared to DNA-PK, has a greater preference for blunt ends and 3' single-base overhangs but a weaker preference for long overhangs, and (2) nicks are effective in attracting and activating PARP. The possible implications of the preferences of PARP and DNA-PK for DNA strand interruptions in vivo are discussed.

The action of Escherichia coli endonuclease III on multiply damaged sites in DNA.

Energy deposition by ionizing radiation can lead to the formation of clustered DNA damage, i.e. more than one lesion situated within a helical turn of DNA. Among the postulated lesions are those characterized by damaged bases and abasic sites on opposite strands. Enzymatic removal of such lesions may inadvertently lead to the formation of double-strand breaks. To test this hypothesis, we have constructed model substrates containing damaged bases (5,6-dihydrothymine) or abasic sites set one, three, five and seven bases apart on opposite strands, and examined the reactivity of Escherichia coli endonuclease III towards these substrates. Endonuclease III demonstrates two activities; as a glycosylase that removes saturated pyrimidine bases, such as dihydrothymine, and as an AP lyase that cleaves DNA strands at abasic sites. Analysis of endonuclease III-treated dihydrothymidine containing plasmid DNA by agarose gel electrophoresis indicated that the enzyme generated only single-strand breaks when the base damage was set one and three base-pairs apart, and only slowly introduced double-strand breaks in the other substrates. Endonuclease III treatment of the abasic site-containing DNA, however, readily yielded double-strand breaks. Taken together, these results indicate that the glycosylase activity of the enzyme, but not the AP lyase activity, is inhibited by the presence of a closely positioned break in the opposite strand.

Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease.

The apurinic/apyrimidinic endonucleases (APE) contain several highly conserved sequence motifs. The glutamic acid residue in a consensus motif, LQE96TK98 in human APE (hAPE-1), is crucial because of its role in coordinating Mg2+, an essential cofactor. Random mutagenesis of the inactive E96A mutant cDNA, followed by phenotypic screening in Escherichia coli, led to isolation of an intragenic suppressor with a second site mutation, K98R. Although the Km of the suppressor mutant was about sixfold higher than that of the wild-type enzyme, their kcat values were similar for AP endonuclease activity. These results suggest that the E96A mutation affects only the DNA-binding step, but not the catalytic step of the enzyme. The 3' DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global change of the protein conformation is induced by the single or double mutations, but a local perturbation in the structural environment of tryptophan residues may be induced by the K98R mutation. The wild-type and suppressor mutant proteins have similar Mg2+ requirement for activity. These results suggest a minor perturbation in conformation of the suppressor mutant enabling an unidentified Asp or Glu residue to substitute for Glu96 in positioning Mg2+ during catalysis. The possibility that Asp70 is such a residue, based on its observed proximity to the metal-binding site in the wild-type protein, was excluded by site-specific mutation studies. It thus appears that another acidic residue coordinates with Mg2+ in the mutant protein. These results suggest a rather flexible conformation of the region surrounding the metal binding site in hAPE-1 which is not obvious from the X-ray crystallographic structure.

Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds.

Association of ATM activation and DNA repair with induced radioresistance after low-dose irradiation.

Mammalian cells often exhibit a hyper-radiosensitivity (HRS) to radiation doses <20 cGy, followed by increased radioresistance (IRR) at slightly higher doses (∼20-30 cGy). Here, the influence of DNA double-strand break repair (DSBR) on IRR was examined. The failure of Ataxia telangiectasia (AT) cells to undergo IRR reported by others was confirmed. Flow cytometric analysis indicated that normal cells fail to show a measurable increase in serine 1981 phosphorylated AT-mutated (ATM) protein after 10 cGy up to 4 h post irradiation, but a two- to fourfold increase after 25 cGy. Similarly, more proficient reduction of phosphorylated histone H2AX was observed 24 h after 25 cGy than after 10 cGy, suggesting that DSBR is more efficient during IRR than HRS. A direct examination of the consequences of inefficient DNA repair per se (as opposed to ATM-mediated signal transduction/cell cycle responses), by determining the clonogenic survival of cells lacking the DNA repair enzyme polynucleotide kinase/phosphatase, indicated that these cells have a response similar to AT cells, i.e. HRS but no IRR, strongly linking IRR to DSBR.

Impairment in Holocaust survivors after 33 years: data from an unbiased community sample.

The authors compared the psychiatric status of 135 survivors of the Holocaust with that of control subjects. This study stands apart from most previous reports in that the two groups were generated by a random sample survey of all heads of households in a community (Montreal). Survivors were more likely to have mild psychiatric symptoms regardless of the age at which they experienced the Holocaust. The difference between survivors and controls in levels of mild psychiatric symptoms was greatly amplified in those respondents who perceived a recent increase in anti-Semitism in Montreal.

Bradycardia: a late complication following cardiac transplantation.

Symptomatic bradycardia occurred in 11 of 213 patients (5.2%) surviving > 1 year after orthotopic heart transplantation, most of whom were managed with permanent pacemakers. Evidence for sinus and/or atrioventricular node dysfunction was present in many cases, but patients at risk for this complication could not be predicted from baseline clinical characteristics.

Removal by human apurinic/apyrimidinic endonuclease 1 (Ape 1) and Escherichia coli exonuclease III of 3'-phosphoglycolates from DNA treated with neocarzinostatin, calicheamicin, and gamma-radiation.

DNA strand breaks with terminal 3'-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4'-carbon of DNA deoxyribose groups. Included among these agents are gamma-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin. However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks. Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents. Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from gamma-irradiated DNA, although there appeared to be a small resistant subpopulation. The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes. These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.

Early outcome of initiating amiodarone for atrial fibrillation in advanced heart failure.

BACKGROUND: Little information exists about the early outcomes of initiating amiodarone for atrial fibrillation in patients with advanced heart failure. This study assessed the initial rate of success and complications of amiodarone therapy initiated for patients with atrial fibrillation during hospitalization for heart failure. METHODS: We reviewed medical records for 37 consecutive patients with left ventricular ejection fractions

Response of base excision repair enzymes to complex DNA lesions.

There is now increasing evidence that ionizing radiation generates complex DNA damage, i.e. two or more lesions--single-strand breaks or modified nucleosides--located within one to two helical turns on the same strand or on opposite strands. Double-strand breaks are the most readily recognizable clustered lesions, but they may constitute a relatively minor fraction of the total. It is anticipated that clustered lesions may play a significant role in cellular response to ionizing radiation since they may present a major challenge to the DNA repair machinery. The degree of lesion complexity increases with increasing LET. This has potential implications for space travel because of exposure to high-LET cosmic radiation. It is therefore critical that we begin to understand the consequences of such damaged sites, including their influence on DNA repair enzymes. This paper presents a short review of our current knowledge of the action of purified DNA repair enzymes belonging to the base excision repair pathway, including DNA glycosylases and apurinic/apyrimidinic endonucleases, on model complex lesions.

Interaction of DNA-dependent protein kinase and poly(ADP-ribose) polymerase with radiation-induced DNA strand breaks.

Two of the enzymes involved in the response of mammalian cells to ionizing radiation are the DNA-dependent protein kinase and poly(ADP-ribose) polymerase. These enzymes are known to be activated by binding to DNA strand breaks, but previous studies designed to look at strand break specificity have employed enzymatically generated strand breaks and not irradiated DNA. Using highly purified DNA-dependent protein kinase, we compared enzyme activation by a series of DNA substrates. Irradiated plasmid DNA activated DNA-dependent protein kinase in a dose-dependent manner. When calculated in terms of the molar concentration of double-strand breaks, the enzyme activation by irradiated DNA was comparable to that by restriction enzyme-cleaved DNA. Linear DNA purified after plasmid irradiation also activated DNA-dependent protein kinase to a comparable extent, but nicked DNA, either isolated from irradiated plasmid or generated by DNase I, failed to activate the enzyme. A comparison of the enzyme activation by plasmid molecules with different 3'- and 5'-terminal groups indicated that the chemical nature of the DNA termini at the double-strand break does not significantly influence the response of the DNA-dependent protein kinase. Similar experiments with poly(ADP-ribose) polymerase demonstrated that single- and double-strand breaks activate this enzyme with almost equal efficiency, but because of their greater number, single-strand breaks dominate the response of poly(ADP-ribose) polymerase to irradiated DNA.

Polybrene/DMSO-assisted gene transfer. Generating stable transfectants with nanogram amounts of DNA.

Polybrene/DMSO-assisted gene transfer is a simple and versatile transfection strategy capable of producing high numbers of stable transfectants from adherent monolayer cultures with low (nanogram) quantities of exogenous DNA. The procedure involves two stages: adsorption and internalization. The former is mediated by polybrene (a polycation polymer) and favors the uniform coating of target cells with polybrene-DNA complexes. Following adsorption, the cells are permeabilized by a brief exposure to dimethyl sulfoxide (DMSO) to facilitate the uptake of DNA complexes. Diverse cell types can be exposed to a wide range of polybrene concentrations without adverse effects. By contrast, the key determinant of success is the DMSO permeabilization regime, which must be configured independently for each cell line. Protocols optimized for gene transfer in murine and human fibroblasts are presented along with a guide for the rapid optimization of the method. The advantages and limitations of the method are also discussed.

Gel microdrop flow cytometry assay for low-dose studies of chemical and radiation cytotoxicity.

Low-level cytotoxicity may affect low-dose dose-response relations for cancer and other endpoints. Conventional colony-forming assays are rarely sensitive enough to examine small changes in cell survival and growth. Automated image-analysis techniques are limited to ca. 10(4) cells/plate. An alternative method involves encapsulation of single proliferating cells into ca. 35-75-microm-diameter agarose gel microdrops (GMDs) that are randomly grouped, differential exposure of these groups, culture at 37 degrees C for 3-5 days, and finally GMD analysis by flow cytometry (FC) to determine the ratio of GMDs containing multiple versus single cells as a measure of clonogenic survival. This GMD/FC assay was used to examine low-dose cell killing induced by a cooked-meat mutagen/rodent-carcinogen (MeIQx) in DNA-repair-deficient/metabolically-sensitive CHO cells. Results of conventional colony-forming assays using up to 30 replicate plates indicate a shouldered, threshold-like dose-response; in contrast, those obtained using the GMD/FC assay suggest "hypersensitivity"-like nonlinearity in dose-response. The GMD/FC assay was also applied to human A549 lung cells after GMD-encapsulation and gamma radiation followed by culture for a total of 4 days, to examine survival after exposure to > or =100 cGy delivered at a relatively low dose rate (0.18 cGy/min). Dose-response for clonogenic growth was again observed to be reduced with apparent nonlinear suggesting hypersensitivity between 0 and 50 cGy, insofar as doses of 5 and 10 cGy appear to be ca. fivefold more effective per unit dose than the 50- or 100-cGy doses used. The GMD/FC assay may thus reveal low-dose dose-response relations for chemical and radiation effects on cell proliferation/killing with implications for low-dose risk assessment.