Rapid Immunoassays for Detection of UV-Induced Cyclobutane Pyrimidine Dimers in Whole Bacterial Cells

Immunoassays were developed to measure DNA damage retained by UV-irradiated whole bacterial cells. Active Mycobacterium parafortuitum and Serratia marcescens cells were fixed and incubated with cyclobutane pyrimidine dimer-binding antibodies after being exposed to known UV doses (254 nm). When both fluorescent (Alexa Fluor 488) and radiolabeled (¹²⁵I) secondary antibodies were used as reporters, indirect whole-cell assays were sensitive enough to measure intracellular UV photoproducts in M. parafortuitum and S. marcescens cells as well as photoenzymatic repair responses in S. marcescens cells. For the same UV dose, fluorescent DNA photoproduct detection limits in whole-cell assays (immunofluorescent microscopy) were similar to those in fluorescent assays performed on membrane-bound DNA extracts (immunoslot blot). With either fluorescent or radiolabeled reporters, the intracellular cyclobutane pyrimidine dimer content of UV-irradiated whole bacterial cells could be reliably quantified after undergoing a <0.5-order-of-magnitude decrease in culturability. Immunofluorescent microscopy results showed that photoenzymatic repair competence is not uniformly distributed among exponential-growth UV-irradiated pure cultures.     

Site-specific strand breaks in RNA produced by (125)I radiodecay

Decay of ¹²⁵I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of ¹²⁵I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3′-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when ¹²⁵I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that ¹²⁵I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules.     

UV-induced repair inEscherichia coli B/r Hcr− thy trp cells: Its dependence on UV damage

Irradiation ofEscherichia coli B/r Hcr^? thy trp cells with a low UV-dose permits a post-replication repair of DNA and decreases the breakdown of DNA after a successive irradiation of cells with high UV doses. The usefulness of a repair function of the protein synthesized after a low irradiation dose increases with the increasing damage of DNA.     

Interaction of RecBCD Enzyme with DNA at Double-Strand Breaks Produced in UV-Irradiated Escherichia coli: Requirement for DNA End Processing

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m²). The activity decrease was seen by an increase in survival of phage T4 2⁻ of about 200-fold (phage T4 2⁻ has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb⁺) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.     

Repair of endogenous DNA base lesions modulate lifespan in mice

The accumulation of DNA damage is thought to contribute to the physiological decay associated with the aging process. Here, we report the results of a large-scale study examining longevity in various mouse models defective in the repair of DNA alkylation damage, or defective in the DNA damage response. We find that the repair of spontaneous DNA damage by alkyladenine DNA glycosylase (Aag/Mpg)-initiated base excision repair and O⁶-methylguanine DNA methyltransferase (Mgmt)-mediated direct reversal contributes to maximum life span in the laboratory mouse. We also uncovered important genetic interactions between Aag, which excises a wide variety of damaged DNA bases, and the DNA damage sensor and signaling protein, Atm. We show that Atm plays a role in mediating survival in the face of both spontaneous and induced DNA damage, and that Aag deficiency not only promotes overall survival, but also alters the tumor spectrum in Atm^−/− mice. Further, the reversal of spontaneous alkylation damage by Mgmt interacts with the DNA mismatch repair pathway to modulate survival and tumor spectrum. Since these aging studies were performed without treatment with DNA damaging agents, our results indicate that the DNA damage that is generated endogenously accumulates with age, and that DNA alkylation repair proteins play a role in influencing longevity.     

RecA Protein Recruits Structural Maintenance of Chromosomes (SMC)-like RecN Protein to DNA Double-strand Breaks

Escherichia coli RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand break (DSB) repair. Previous studies show that GFP-RecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the nucleoid is unknown. Here, we show that the assembly of GFP-RecN foci on the nucleoid in response to DNA damage involves a functional interaction between RecN and RecA. A novel RecA allele identified in this work, recA^Q300R, is proficient in SOS induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage. Cells carrying recA^Q300R fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The ATPase-deficient RecN^K35A binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair and that it is required upstream of RecA-mediated strand exchange.     

Neurons Efficiently Repair Glutamate-induced Oxidative DNA Damage by a Process Involving CREB-mediated Up-regulation of Apurinic Endonuclease 1

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca²⁺ influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca²⁺ chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca²⁺/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca²⁺- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca²⁺-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.     

The Listeria monocytogenesiap gene as an indicator gene for the study of PrfA-dependent regulation

The Bacillus subtilis 168 RecR protein bound to duplex DNA in the presence of ATP and divalent cations (Mg²⁺ and Zn²⁺) was visualized by electron microscopy as a nearly spherical particle. A RecR homomultimer is frequently located at the intersection of two duplex DNA strands in an interwound DNA molecule, generating DNA loops of variable length. Two individual DNA molecules bound to the same protein are seen at a very low frequency, if at all. The association of RecR with the intersection of two duplex DNA strands is more often seen in supercoiled than with relaxed or linear DNA. The RecR protein displays a slight but significant preference for negatively supercoiled over linear DNA. The minimum substrate size for RecR protein is about 150 bp in length. A possible mechanism for RecR function in DNA repair is discussed.     

Restriction Enzyme Digests of Rapidly Renaturing Fragments of Vaccinia Virus DNA

Vaccinia virus DNA fragments that have been denatured by alkali and then neutralized contain a fraction that rapidly reforms duplex structures. The fraction is enriched by fractionating on hydroxyapatite columns and serves as a substrate for digestion by two restriction endonucleases isolated from Hemophilus parainfluenzae, Hpa I and HPa II. The patterns obtained by gel electrophoresis of the digested fragments show the presence of three major bands after Hpa I digestion and four major bands after Hpa II digestion. The DNA that is isolated from some of these bands quickly reforms duplex regions after alkaline denaturation. The size of the DNA segments in the major bands has been estimated to be in the range of 0.44 × 10⁶ to 3.2 × 10⁶ daltons. The fragments which rapidly reform duplex chains after denaturation are sensitive to single-strand-specific nucleases. These results are consistent with a model of vaccinia virus DNA which has a covalent link connecting complementary chains.     

Identification of the DNA damage-responsive elements of therhp51 + gene,arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51 ⁺ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51 ⁺ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51 ⁺ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51 ⁺ expression.     

A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage

The molecular role of poly (ADP-ribose) polymerase-1 in DNA repair is unclear. Here, we show that the single-strand break repair protein XRCC1 is rapidly assembled into discrete nuclear foci after oxidative DNA damage at sites of poly (ADP-ribose) synthesis. Poly (ADP-ribose) synthesis peaks during a 10 min treatment with H₂O₂ and the appearance of XRCC1 foci peaks shortly afterwards. Both sites of poly (ADP-ribose) and XRCC1 foci decrease to background levels during subsequent incubation in drug-free medium, consistent with the rapidity of the single-strand break repair process. The formation of XRCC1 foci at sites of poly (ADP-ribose) was greatly reduced by mutation of the XRCC1 BRCT I domain that physically interacts with PARP-1. Moreover, we failed to detect XRCC1 foci in Adprt1^–/– MEFs after treatment with H₂O₂. These data demonstrate that PARP-1 is required for the assembly or stability of XRCC1 nuclear foci after oxidative DNA damage and suggest that the formation of these foci is mediated via interaction with poly (ADP-ribose). These results support a model in which the rapid activation of PARP-1 at sites of DNA strand breakage facilitates DNA repair by recruiting the molecular scaffold protein, XRCC1.     

Estimation of DNA, RNA and 125 I- and 3 H-labelled DNA in the same sample

Estimation of DNA, RNA, and the specific activity of DNA after labelling with [³H]thymidine and/or [¹²⁵I]iodeoxyuridine has been accomplished using a recently developed procedure for the estimation of DNA with p-nitrophenylhydrazine (pNPH). Samples of the pNPH reaction mixture are used for RNA estimation by th orcinol procedure and for ¹²⁵I and tritium measurement. Correction for ¹²⁵I contribution to the tritium measurement in double labelling experiments is accomplished either by use of a simple calibration curve (for high ${}^3\text{H}{}^{125}\text{I}$ ratios) or by removal of ¹²⁵I by hydrolysis and precipitation as AgI; in the latter procedure the efficiency of removal of ¹²⁵I was greater than 99%.     

The role of DNA polymerases in DNA repair in Escherichia coli: DNA polymerase I-dependent repair following chemical alkylation of permeabilized bacteria.

Many mutagens and carcinogens damage DNA and elicit repair synthesis in cells. In the present study we report that alkylation of the DNA of Escherichia coli that have been made permeable to nucleotides by toluene treatment results in the expression of a DNA polymerase I-directed repair synthesis. The advantage of the system described here is that it permits measurement of only DNA polymerase I-directed repair synthesis and serves as a simple, rapid method for determining the ability of a given chemical to elicit “excision-repair” in bacteria.DNA ligation is intentionally prevented in our system by addition of the inhibitor nicotinamide mononucleotide. In the absence of DNA ligase activity, nick translation is extensive and an “exaggerated” repair synthesis occurs. This amplification of repair synthesis is unique for DNA polymerase I since it is not observed in mutant cells deficient in this polymerase. DNA ligase apparently controls the extent of nucleotide replacement by this repair enzyme through its ability to rejoin “nicks” thereby terminating the DNA elongation process.The nitrosoamides N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea, as well as the nitrosoamidines N-methyl-N′-nitro-N-nitrosoguanidine and N-ethyl-N′-nitro-N-nitrosoguanidine, elicit DNA polymerase I-directed repair synthesis. Methyl methanesulphonate is especially potent in this regard, while its ethyl derivative, ethyl methanesulphonate, is a poor inducer of DNA polymerase I activity in permeabilized cells.     

The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence

Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA–protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition.     

Molecular analysis of base damage clustering associated with a site-specific radiation-induced DNA double-strand break

Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted (125)I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the (125)I-targeted base in the DNA duplex. An increased yield of base damage (purine > pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating that the damage detected proximal to the DSB was produced by the (125)I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the approximately 32-bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the (125)I-decay site that may form via a charge migration mechanism.     

HDF1 and RAD17 Genes are Involved in DNA Double-strand Break Repair in Stationary Phase Saccharomyces cerevisiae

DNA repair, checkpoint pathways and protection mechanisms against different types of perturbations are critical factors for the prevention of genomic instability. The aim of the present work was to analyze the roles of RAD17 and HDF1 gene products during the late stationary phase, in haploid and diploid yeast cells upon gamma irradiation. The checkpoint protein, Rad17, is a component of a PCNA-like complex—the Rad17/Mec3/Ddc1 clamp—acting as a damage sensor; this protein is also involved in double-strand break (DBS) repair in cycling cells. The HDF1 gene product is a key component of the non-homologous end-joining pathway (NHEJ). Diploid and haploid rad17Δ/rad17Δ, and hdf1Δ Saccharomyces cerevisiae mutant strains and corresponding isogenic wild types were used in the present study. Yeast cells were grown in standard liquid nutrient medium, and maintained at 30°C for 21 days in the stationary phase, without added nutrients. Cell samples were irradiated with ⁶⁰Co γ rays at 5 Gy/s, 50 Gy ≤ Dabs ≤ 200 Gy. Thereafter, cells were incubated in PBS (liquid holding: LH, 0 ≤ t ≤ 24 h). DNA chromosomal analysis (by pulsed-field electrophoresis), and surviving fractions were determined as a function of absorbed doses, either immediately after irradiation or after LH. Our results demonstrated that the proteins Rad17, as well as Hdf1, play essential roles in DBS repair and survival after gamma irradiation in the late stationary phase and upon nutrient stress (LH after irradiation). In haploid cells, the main pathway is NHEJ. In the diploid state, the induction of LH recovery requires the function of Rad17. Results are compatible with the action of a network of DBS repair pathways expressed upon different ploidies, and different magnitudes of DNA damage.     

PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1^−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1^−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.     

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.