Top PDF Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways

Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways

Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways

E. coli endonuclease III (EndoIII) is a DNA glycosylase that excises oxi- dized pyrimidines from DNA, functioning as part of the base excision repair (BER) pathway in order to maintain the integrity of the genome [1]. EndoIII contains a [4Fe4S] 2+ cluster that is relatively insensitive to reduction and oxidation in solution [2]; as a result, it was initially proposed that the cluster served only a structural role within the protein. MutY is another E. coli BER glycosylase, homologous to EndoIII, that also contains a [4Fe4S] 2+ cluster [3]. MutY, found in organisms from bacteria to man, is involved in the repair of oxoG:A mismatches [4]; in humans, inherited defects in MUTYH are associated with a familial form of colon cancer known as MUTYH-associated polyposis (MAP) and many MAP-associated vari- ants are localized near the [4Fe4S] cluster [4]. Furthermore, in the case of MutY, it has been shown that the cluster is not required for folding or stability [3], or di- rect participation in the intrinsic glycosidic bond hydrolysis catalysis [5], making the widespread presence of conserved, noncatalytic [4Fe4S] clusters difficult to explain. Notably, the earliest studies with EndoIII and MutY looked only at free protein in solution, neglecting the effect of DNA binding on redox potential. Experiments carried out on DNA-modified electrodes have demonstrated that, in both EndoIII and MutY, the cluster undergoes a negative shift in potential associated with binding to the DNA polyanion and is activated toward reversible redox activity [6]. In these experiments, DNA monolayers were formed on gold electrodes, and upon addition of EndoIII or MutY, a reversible signal with a midpoint potential ranging from 60 to 95 mV versus NHE was observed. Importantly, the introduction of just a single mismatch or abasic site into DNA led to signal attenuation, showing that electron transfer between the protein and the electrode was through the π -stacked base pairs in a process known as DNA-mediated charge transport (DNA CT) [7]. In this process, charge is funneled from the electrode surface through the π -stack of the DNA bases to reach the redox probe (a protein in this case); the only requirement is that the probe must be electronically coupled to the DNA π -stack. Remarkably, the sensitivity to base stacking observed with EndoIII and MutY was comparable to that obtained using small molecules such as Nile blue or methylene blue that intercalate directly into the base stack.
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Expanding the Repertoire of DNA-Mediated Signaling in DNA Repair

Expanding the Repertoire of DNA-Mediated Signaling in DNA Repair

DNA charge transfer (CT) is the phenomenon by which electrons are transferred through the π-stacked bases of DNA (1). This process is exquisitely sensitive, as perturbations to individual base pairs alter the geometry of the π-stack dramatically, thereby attenuating the conductivity of the DNA “wire.” However, nicks in the sugar phosphate backbone do not affect this process (2). Double-stranded DNA in a variety of conformations, including G-quadruplexes and Holliday junctions, has been shown to conduct charge (3-6). Recently, a model was proposed whereby redox-active proteins utilize DNA-mediated CT to localize near the site of DNA lesions because of the attenuation of CT at these sites. In the model system, MutY and EndoIII, two base excision repair (BER) proteins from E. coli, are activated towards oxidation upon DNA binding. Protein oxidation releases an electron to a distally bound protein, which, upon reduction, dissociates from the DNA. However, if an intervening lesion is present, this electron transport process can no longer occur, and the repair proteins remain bound to the DNA and eventually progress towards the lesion (7-10).
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DNA Mediated Charge Transport Signaling Within the Cell

DNA Mediated Charge Transport Signaling Within the Cell

Taken together, these results provide even stronger evidence that DNA-mediated signaling is used as a general mechanism by which DNA repair proteins with 4Fe-4S clusters locate damage within E. coli. Here, upon spectroscopically showing that UvrC contains a 4Fe-4S cluster, DNA-modified electrochemistry was used to establish that UvrC shares a DNA-bound redox potential with MutY, EndoIII, and DinG. Previous work established that DNA-mediated signaling may be utilized by EndoIII, MutY, and DinG to coordinate the search and repair process. Here we show results that expand this putative DNA-mediated signaling network to include UvrC, the fourth DNA-processing enzyme shown to contain a 4Fe-4S cluster within E. coli. Regardless of the fact that the proteins within this network are from different repair pathways, target different substrates, and perform different catalytic functions, the proteins rely upon one another to maintain the fidelity of the genome, especially under stress. The unifying factor of this network of DNA- processing enzymes is that they contain 4Fe-4S clusters that share a redox potential. It will be of great interest to see if the trends observed in E. coli also hold true in higher organisms. DNA-mediated charge transport chemistry offers an elegant mechanism to report on the integrity of the DNA duplex. DNA CT has been shown to travel long distances, is fast, and is highly sensitive to perturbations to base-stacking. This work adds to a growing body of evidence that indicates that proteins with 4Fe-4S cluster not only can participate in long-range redox reactions via DNA CT, but also use this chemistry as a means of cooperative signaling to coordinate the efficient repair and processing of DNA within cells.
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DNA Mediated Charge Transport in DNA Repair

DNA Mediated Charge Transport in DNA Repair

Though the E. coli rnf genes have not been biochemically characterized, it has been demonstrated that inactivation of these genes has an effect on SoxR mediated soxS expression (25). The soxRS system senses oxidative stress and activates transcription of a wide variety of genes to protect against and repair oxidative damage (interestingly, one of the genes targeted is yggX (19)) (26). Activation of the soxRS regulon is mediated by SoxR, a [2Fe2S] cluster transcription factor (27-29). Upon oxidation of the cluster in SoxR from the 1+ to the 2+ state, transcription of soxS is initiated. SoxS transcription is transient; within minutes after administration of oxidants has ceased, SoxR is rereduced and soxS is no longer transcribed (29). The pathways for oxidation and rereduction of SoxR are not fully understood, though SoxR is activated within the cell by administration of paraquat (29) and it has been demonstrated in vitro that SoxR can be oxidized from a distance, in a DNA-mediated fashion, by guanine radicals or electrochemical methods (30, 31). Inactivation of the E. coli rnf genes slows the deactivation of soxS expression, indicating that the rnf gene products may be involved in the rereduction of SoxR (25).
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Involvement of the Yeast DNA Polymerase δ in DNA Repair in Vivo

Involvement of the Yeast DNA Polymerase δ in DNA Repair in Vivo

Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, [r]

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Promoter  DNA  Methylation  of  DNA  Repair  Genes  in  Cancer

Promoter DNA Methylation of DNA Repair Genes in Cancer

way, a C-to-T point mutational event occurs. Because thymine is a normal component of human dnA, this mutation may not be correctly recognized by the dnA repair mechanisms. Instead of repairing the mutated thymine, the complementary strand guanine may be substituted for adenine to form the normal T–A opposition. Hence, a G-to-A point mutation occurs. The transformation of C- to- T can occur either through spontaneous deamination of 5mC or by an enzyme-mediated mechanism where methyltransferase binding results in deamination before the methyl transfer to form uracil, which is then substituted by thymine after two rounds of dnA replication. The major cause of the high mutation rate at Cpg dinucleotides is likely to be spontaneous deamination of 5mC. 58
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DNA methylation reprogramming and DNA repair in the mouse zygote

DNA methylation reprogramming and DNA repair in the mouse zygote

So far, there are no clear evidences for the existence of a bona fide demethylase, which catalyzes the direct removal of the methyl group. Several candidates have been proposed, but none could be verified as a global demethylase. MBD2 was shown to demethylate DNA directly (Bhattacharya et al., 1999), but this data could not been reproduced by the others (Ng et al., 1999). In Escherichia coli the dioxygenases AlkA and AlkB are able to directly demethylate 3-methylcytosine (3mC) and their human homologs ABH2 and ABH3 were identified (Duncan et al., 2002). Due to more stable C-C bond in 5mC compared to the less stable C-N bond in 3mC, it is biochemically unlikely that a dioxygenase directly demethylates 5mC. Nevertheless, the discovery of 5- hydroxymethyl-cytidine (5hmC) in mammalian DNA (Kriaucionis and Heintz, 2009; Tahiliani et al., 2009) and corresponding dioxygenases TET1, TET2 and TET3 catalyzing the synthesis of 5hmC suggests a possible candidate mechanism for bona fide demethylation. Liutkeviciute et al. have shown that DNA methyltransferase DNMT1 is able to directly remove the hydroxymethyl group in vitro in absence of SAM (Liutkeviciute et al., 2009), implying 5hmC as an intermediate compound in the direct DNA demethylation. 5hmC could also be the target for further enzymatic oxidation, which would lead to enzymatic de- carboxylation and yielding the unmodified cytosine. The recent study by Ito et al. has demonstrated the presence of TET1 in mouse zygotes and thus providing the potential evidences of 5hmC presence in preimplantation embryos (Ito et al., 2010).
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DNA repair and the cytotoxic effects of cisplatin and DNA thiobases

DNA repair and the cytotoxic effects of cisplatin and DNA thiobases

The chemistry o f cisplatin and how it interacts with DNA has been extensively reviewed (Jamieson and Lippard, 1999; Trimmer and Essigmann, 1999). Cisplatin shows significant activity against a wide variety o f tumours including ovary, bladder, lung, and head and neck and is a comm on com ponent o f m any chem otherapy regimes. It has been used most successfully in the treatment o f testicular carcinomas w ith greater than 90% now curable by this drug. The structure o f cisplatin, carboplatin and the inactive isomer transplatin are illustrated in Fig 1.12^. Uptake o f cisplatin into cells is suggested to occur via passive diffusion. In the bloodstream, due to the high concentration o f Cl' ions (-IGOmM), cisplatin is relatively unreactive. However, in the cell the Cl' concentration is much lower (~ 4mM) and this facilitates the hydrolysis o f the two Cl ligands on the cisplatin molecule. W ater molecules displace the two Cl ligands in a stepwise fashion to generate an aquated positively charged complex (Fig 1.125). As w ater is a good leaving group, the aquated cisplatin com plex can react w ith cellular nucleophiles including DNA, RNA, proteins and cellular thiols (e.g. glutathione and metallothionein). DNA is suggested to be the important target by which cisplatin exerts its cytotoxic effect as E. coli, yeast and mammalian cells defective in the repair o f cisplatin induced DNA damage are hypersensitive to killing by this drug. The major DNA target is the N^-position o f purines. The major adducts induced by cisplatin are illustrated in Fig 1.12C and their relative abundance in Table 1.1.
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Redox signaling in colonial hydroids: many pathways for peroxide

Redox signaling in colonial hydroids: many pathways for peroxide

On the basis of these data, we hypothesize that in hydroid colonies ROS participate in a number of putative signaling pathways. High levels of ROS may be a factor in the cell and tissue death that seem to affect peripheral stolon tips when the environment is rapidly changing. Such a process would seem adaptive – if the colony becomes ‘overextended,’ stolons can retreat and the nutrients in the cells and tissues of the stolon may be taken up by the remainder of the colony. ROS emitted from the colony also seem to have an extra-colony function, perhaps in suppressing the growth of bacteria or other parasites. Hydractiniid hydroid colonies grow on snail shells that are crowded with epifauna and probably some of these can be rebuffed by peroxide. Notably, the foot region of Hydra is characterized by the activity of a peroxidase (Hoffmeister- Ullerich et al., 2002). Hydra may also emit peroxide and may use this peroxidase to protect its own tissue at the point of attachment to the substratum. More moderate levels of ROS in stolon tips seem to act as a growth factor, triggering outward growth, inhibiting branching and, possibly, mediating the redox signaling emanating from mitochondrion-rich EMCs. Treatment with exogenous peroxide suggests that stolon tips are capable of concentrating peroxide. Peroxide emitted from polyp–stolon junctions could be carried by gastrovascular flow to stolon tips. Nevertheless, because of the multiple pathways for peroxide, the particular phenotypic effects may depend on the spatial and temporal patterns of ROS formation within the colony. While the work reported here serves to outline the broad possibilities for signaling using ROS in colonial hydroids, considerable amounts of future research will be required to elucidate these spatial and temporal patterns, as well as the molecular targets of ROS.
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Mechanism of DNA loading by the DNA repair helicase XPD

Mechanism of DNA loading by the DNA repair helicase XPD

These modified enzymes were assessed for DNA bind- ing, ATPase and helicase activity (Figure 5A, B and C, re- spectively). The DNA binding affinities of the crosslinked species were not significantly different from the non- crosslinked controls (Figure 5A, Table 2), however DNA- stimulated ATPase activity of TaXPD was reduced by 2–3- fold (Figure 5B, Table 2). Helicase activity increased with the protein concentration up to 1 !M TaXPD (Supple- mentary Figure S9A). The time courses displayed clear lag phases, which suggests that TaXPD may proceed through multiple repeated steps to fully unwind the duplex DNA, as observed for other helicases (42). The intra-molecular crosslinked mutants, 100C-238C and 107C-312C, showed a large reduction (but not abolition) of the helicase activity (Figure 5C and Supplementary Figure S9A). The decrease in the helicase activity of 100C-238C appeared proportional with the degree of crosslinking (Figure 5C). Both biotin- modified DNA and neutravidin biotin-DNA (which is too big to pass through any conceivable pore) complex behaved normally in the helicase assay, ruling out DNA threading as a model (Supplementary Figure S9B).
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Saturation of DNA Mismatch Repair and Error Catastrophe by a Base Analogue in Escherichia coli

Saturation of DNA Mismatch Repair and Error Catastrophe by a Base Analogue in Escherichia coli

Deoxyribosyl-dihydropyrimido[4,5-c][1,2]oxazin-7-one (dP) is a potent mutagenic deoxycytidine-derived base analogue capable of pairing with both A and G, thereby causing G · C → A · T and A · T → G · C transition mutations. We have found that the Escherichia coli DNA mismatch-repair system can protect cells against this mutagenic action. At a low dose, dP is much more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain. At higher doses, the difference between the wild- type and the mutator strains becomes small, indicative of saturation of mismatch repair. Introduction of a plasmid containing the E. coli mutL ⫹ gene significantly reduces dP-induced mutagenesis. Together, the results indicate that the mismatch-repair system can remove dP-induced replication errors, but that its capacity to remove dP-containing mismatches can readily be saturated. When cells are cultured at high dP concentration, mutant frequencies reach exceptionally high levels and viable cell counts are reduced. The observations are consistent with a hypothesis in which dP-induced cell killing and growth impairment result from excess mutations (error catastrophe), as previously observed spontaneously in proofreading- deficient mutD (dnaQ) strains.
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Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken

DT40 cell-lines.

Impact of DNA repair pathways on the cytotoxicity of piperlongumine in chicken DT40 cell-lines.

of piperlongumine, a panel of DNA repair-deficient cell lines derived from chicken DT40 cells was studied. Our results show that piperlongumine selectively kills cell lines with a defect in homologous recombination (HR). Piperlongumine displays little or no toxicity to cell lines with a defect in other DNA repair pathways, including the base excision repair (BER) that is a major pathway to repair ROS-induced DNA lesions. A deletion of 53BP1 or Ku70 in BRCA1-deficient cell lines restores resistance to piperlongumine, strongly implicating that piperlongumine exerts its cytotoxicity by generating double strand breaks. Unexpectedly, we also discovered that piperlongumine suppresses HR. Altogether, we described the novel mechanisms of cytotoxicity by piperlongumine.
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REV1 and DNA polymerase zeta in DNA interstrand crosslink repair

REV1 and DNA polymerase zeta in DNA interstrand crosslink repair

All eukaryotic Y-family polymerases including REV1 possess ubiquitin-binding motifs (UBM) or ubiquitin-bind- ing zinc finger (UBZ) domains that increase their affinity for ubiquitinated PCNA [Kannouche et al., 2004; Wata- nabe et al., 2004; Bienko et al., 2005; Plosky et al., 2006; Acharya et al., 2008; Sabbioneda et al., 2008; Bomar et al., 2010]. Although the ubiquitination of PCNA is nec- essary for TLS in yeast, recent studies suggest that alter- native pathways may regulate polymerase switching in vertebrates. Analysis of the replication of damaged DNA in chicken DT40 cells demonstrated a predominant role for PCNA ubiquitination in promoting the filling in of postreplication gaps [Edmunds et al., 2008]. However, REV1-dependent TLS across a TT 6-4 photoproduct (a dominant UV lesion) in DT40 cells carrying a PCNA K164 mutation appears to be normal as measured by a plasmid system [Szu¨ts et al., 2008]. Recent work from the Livneh group used mouse embryonic fibroblasts in which specific TLS genes associated with ubiquitination of PCNA were manipulated. These studies showed that elim- inating expression of REV3, Polh, or REV1 in PCNA K164R/K164R mouse embryo fibroblasts further increased their sensitivity to UV-radiation indicating the existence of a TLS pathway that is independent of PCNA ubiquitination [Hendel et al., 2011]. At least in DT40 cells, this noncanonical TLS pathway appears to be largely dependent on REV1 [Edmunds et al., 2008; Szu¨ts et al., 2008]. Recent studies identified the FA core com- plex as being an important regulator of REV1 localization to stalled replication forks resulting from UV or cisplatin treatment [Mirchandani et al., 2008; Hicks et al., 2010; Kim et al., 2012].
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Efficient Repair of DNA Breaks in Drosophila: Evidence for Single-Strand Annealing and Competition With Other Repair Pathways

Efficient Repair of DNA Breaks in Drosophila: Evidence for Single-Strand Annealing and Competition With Other Repair Pathways

the larger duplication (Table 2, B vs. C). Apparently tion will lead to mismatches in this heteroduplex, which SSA is only moderately efficient when presented with a are often repaired by the cell’s mismatch repair (MMR) duplication of 158 bp. system. Several studies (Holmes et al. 1990; Carroll et This assay underestimates the rate of w ⴙ loss at the al. 1994; Deng and Nickoloff 1994; Lehman et al. 1994; original site: It is expected that some of the P excision Miller et al. 1997; Taghian et al. 1998) indicate that events will coincide with reinsertion of the element else- the MMR process has a strong bias such that the repaired where in the genome. Most new insertions will express product tends to reflect the sequence of whichever allele w ⫹ , thus masking the loss of P {w ⫹ } at 50C. This process lies farthest from any nick. For experiment C, where we will result in an underestimate of the SSA frequency. hypothesize that virtually all repair events occur by SSA, We can assess the magnitude of the underestimate by we expect a bias in favor of the A form at site ␣ and analyzing loss of P {w ⫹ } at the original site within a sample the C form at site ␤. Furthermore, we expect this bias of the w ⫹ progeny. Out of a total of 413 w ⫹ flies from to be more pronounced in the case of site ␣, since it experiment B, a sample of 79 independent events was lies much closer to a duplication boundary and there- analyzed by PCR, testing for both of the chromosome/ fore closer to a nick in the proposed heteroduplex inter- P-element junctions. After correcting for sampling bias mediate. The results (Figure 4, experiment C) are in (see materials and methods), we calculate that 12.4% good agreement with this expectation, considering that of the w ⫹ progeny from this experiment had precisely 92.6% (41.5 ⫹ 51.1%) of the ␣ sites and 58.5% (51.1 ⫹ lost P {w ⫹ } at the original site. Given that w ⫹ flies were 7.4%) of the ␤ sites have the favored sequence. Experi- 40.5% of the total progeny scored, ⵑ5% of the total ment E also showed a strong bias for the A form at site had undergone precise P {w ⫹ }50C loss with a new P{w ⫹ } ␣ (90%), whereas the two forms were nearly equal at insertion elsewhere. Therefore the true rate of precise site ␤. A similar bias was clear in experiment B, where loss is 53.2% ⫹ 5% ⫽ 58.2%. This analysis confirmed only the ␣ site was duplicated. The favored form (␣A) that the purely phenotypic assay underestimates the rate occurred in 88.5% of the repair products (Figure 4). of precise loss, but that the magnitude of the error is
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Escherichia coli mutator mutD5 is defective in the mutHLS pathway of DNA mismatch repair.

Escherichia coli mutator mutD5 is defective in the mutHLS pathway of DNA mismatch repair.

Competent cells derived from the mutD5 strain are severely affected in their ability to perform mismatch repair, yielding a percentage of mixed bursts close to that of the mu[r]

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MUTANTS OF ESCHERICHIA COLI K-12 DEFECTIVE IN DNA REPAIR AND IN GENETIC RECOMBINATION

MUTANTS OF ESCHERICHIA COLI K-12 DEFECTIVE IN DNA REPAIR AND IN GENETIC RECOMBINATION

HOWARD-FLANDERS 1964). Figure 3 shows the fraction of UV-irradiated T1 phage able to form plaques when plated on the various mutants and the original strain, as a function of [r]

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TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

TDP2 promotes repair of topoisomerase I-mediated DNA damage in the absence of TDP1

expected, Tdp1 / DT40 cells accumulated 3-fold more DNA strand breaks than wild-type cells, following incu- bation with the Top1 poison CPT (Figure 2d). Tdp2 // cells also accumulated higher levels of DNA breaks than did wild-type DT40 cells, though this difference was not statistically significant (P = 0.92; student’s t-test). Importantly, Tdp1 / /Tdp2 // cells accumulated signifi- cantly higher DNA breaks than Tdp1 / cells, suggesting that Tdp2 contributes to the repair of Top1 damage in DT40 cells in the absence of Tdp1 (Figure 2d). Indeed, ectopic expression of hTDP2 in Tdp1 / /Tdp2 // cells reduced the accumulation of DNA strand breaks induced by CPT below that observed in Tdp1 / cells (Figure 3a and b). Furthermore, Tdp1 / /Tdp2 // cells were more sensitive than Tdp1 / or Tdp2 // cells to Top1 breaks induced by CPT (Figure 3c), but not more sensitive to DNA breaks induced by g-radiation (Supplementary Figure S3). Similar results were observed for the anti-cancer Top1 poisons NSC 724998, NSC 725776 or MJ-III-65 (1) as measured by viability assays (Supplementary Figure S4). In contrast, the reverse was not true since deletion of Tdp1 did not further sensitise Tdp2 // cells to the Top2 poison etoposide, suggesting that Tdp1 is unable to contribute significantly to 5 0 -TDP
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DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

Rad26 and Rad28: Genes specifically involved in TC-NER were first identified in humans, where their loss is responsible for Cockayne syndrome (CS). CSA- or CSB-deficient cells are very sensitive to UV, a property that allowed cloning of the human CSA and CSB genes (Troelstra et al. 1992; Henning et al. 1995). Based on sequence homology to the encoded proteins, yeast homologs were identified and the corre- sponding genes were named RAD28 and RAD26, respec- tively (Van Gool et al. 1994; Bhatia et al. 1996). CSB and Rad26 share strong sequence homology, which includes the seven conserved motifs of DNA/RNA helicases in the SNF2 subfamily. Although both proteins exhibit DNA-dependent ATPase activity, neither has detectable helicase activity (Guzder et al. 1996a). In the case of CSB, the ATPase activity is important for in vivo function (Citterio et al. 1998). CSA/ Rad28 is a WD40 repeat protein with no identified catalytic activity and is probably involved in protein interactions (Henning et al. 1995; Bhatia et al. 1996). In contrast to human CS cells, yeast strains lacking either Rad26 or Rad28 are not UV sensitive, which explains why the corre- sponding genes were not recovered in early mutant screens. Analysis of strand-specific repair of CPDs demonstrated that repair of the TS is significantly delayed in rad26D mutants (Van Gool et al. 1994), but is not affected in rad28D cells (Bhatia et al. 1996). Although not evident in a rad26D single mutant, an effect of Rad26 on survival after UV irra- diation can be observed in the absence of GG-NER, with a rad16D rad26D double mutant being more UV sensitive than a rad16D single mutant (Bhatia et al. 1996; Verhage et al. 1996). A rad16D rad26D double mutant, however, is still less UV sensitive than a completely NER-deficient strain such as rad14D, suggesting residual repair of CPDs on the TS by a Rad26-independent TC-NER subpathway (Verhage et al. 1996).
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The role and clinical significance of DNA damage response and repair pathways in primary brain tumors

The role and clinical significance of DNA damage response and repair pathways in primary brain tumors

Given the replication demands in proliferating tumor cells, the HR pathway, which functions during S/G2, may be a valuable target for new and high-therapeutic index GBM treatment regimens. DNA single-strand breaks (SSBs) can lead to DSBs at the replication fork. Unrepaired DSBs are lethal to proliferating cells. Dys- function in the repair of both SSBs and DSBs would be synthetically lethal. The enzyme poly(ADP-ribose) poly- merase 1 (PARP1) plays a key role in the repair of SSBs, [26] while the tumor suppressor BRCA1 is essential for HR-mediated repair of DSBs [27,28]. PARP1 inhibitors, including Olaparib, target cancers which are deficient in the repair of DSBs, exhibit up to 1,000-fold selectivity in killing BRCA1-mutated (DSB-repair deficient) cells, and provide an overall survival and progression-free survival benefit with minimal toxicity in patients with BRCA1- deficient familial breast cancer [29-33]. Unfortunately, the majorities of patients who develop sporadic tumors including malignant gliomas carry wild-type (wt) BRCA1 and are proficient in DSB repair, precluding them from this potent avenue of therapy [34]. We have previously shown that transiently exporting wt-BRCA1 protein from the nucleus (where DSBs are repaired) to the cyto- sol (where apoptosis is activated) makes cancer cells de- fective in the repair of DSBs [35]. We propose to develop an innovative therapeutic strategy that uses this export of BRCA1 from nucleus to cytoplasm to transi- ently convert BRCA1-proficient GBM cells into func- tionally BRCA1-deficient cells and thereby render them susceptible to PARP inhibitor–induced cell killing (Figure 1).
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Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair.

Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair.

Our results suggest that viral DNA integration frequency may dramatically increase in HBV-infected patients when hepatocytes are placed under oxidative stress. Such circum- stances may commonly occur while chronically HBV-infected patients undergo fluctuations in HBV replication levels and serum aminotransferases during disease exacerbations (30). Hepadnaviral DNA integration frequency may even increase when virus replication resumes after periods of antiviral ther- apy. The detection of HBV DNA integrations in cirrhotic liver nodules illustrates that integrations occur during persistent infection and that hepatocytes are clonally amplified in certain persistent infection settings (42). Multiple WHV and HBV DNA integrations in nonclonal liver tissue can be demon- strated in a substantial fraction of long-term chronic carriers (34, 43). HBV DNA integrations can also occur within the host genome early after infection, varying dramatically in their fre- quency (6, 21, 25, 56). The structure of those integrations, which are believed to represent “primary” integration events, closely resembles the DHBV DNA integrations which occur in LMH-D2 cells (16), with their junctions pref- erentially located in the DR1 region. Our data suggest that toxic oxygen radicals may promote hepadnaviral DNA integra- tion in situations such as those described above.
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