Induction of the alternative lengthening of telomeres pathway by trapping of proteins on DNA.

Telomere maintenance is a hallmark of malignant cells and allows cancers to divide indefinitely. In some cancers, this is achieved through the alternative lengthening of telomeres (ALT) pathway. Whilst loss of ATRX is a near universal feature of ALT-cancers, it is insufficient in isolation. As such, other cellular events must be necessary - but the exact nature of the secondary events has remained elusive. Here, we report that trapping of proteins (such as TOP1, TOP2A and PARP1) on DNA leads to ALT induction in cells lacking ATRX. We demonstrate that protein-trapping chemotherapeutic agents, such as etoposide, camptothecin and talazoparib, induce ALT markers specifically in ATRX-null cells. Further, we show that treatment with G4-stabilising drugs cause an increase in trapped TOP2A levels which leads to ALT induction in ATRX-null cells. This process is MUS81-endonuclease and break-induced replication dependent, suggesting that protein trapping leads to replication fork stalling, with these forks being aberrantly processed in the absence of ATRX. Finally, we show ALT-positive cells harbour a higher load of genome-wide trapped proteins, such as TOP1, and knockdown of TOP1 reduced ALT activity. Taken together, these findings suggest that protein trapping is a fundamental driving force behind ALT-biology in ATRX-deficient malignancies.

A key feature of all cancer cells is their ability to divide indefinitely, and this is dependent on circumvention of telomere shortening through induction of a telomere maintenance mechanism, such as the telomerase-independent, Alternative Lengthening of Telomeres (ALT) pathway. The ALT pathway is characterised by loss of the ATRX chromatin remodeler. The current study provides evidence that, in the absence of ATRX, increased trapping of proteins on DNA leads to replication fork stalling and collapse. At telomeres, this leads to ALT pathway activity. These results help to better understand ALT tumours and might, eventually, be instrumental in developing new therapeutic strategies.

Interplay between base excision repair protein XRCC1 and ALDH2 predicts overall survival in lung and liver cancer patients.

BACKGROUND: To deliver efficacious personalised cancer treatment, it is essential to characterise the cellular metabolism as well as the genetic stability of individual tumours. In this study, we describe a new axis between DNA repair and detoxification of aldehyde derivatives with important implications for patient prognosis and treatment. METHODS: Western blot and qPCR analyses were performed in relevant non-transformed and cancer cell lines from lung and liver tissue origin in combination with bioinformatics data mining of The Cancer Genome Atlas database from lung and hepatocellular cancer patients. RESULTS: Using both biochemical and bioinformatics approaches, we revealed an association between the levels of expression of the aldehyde detoxifying enzyme aldehyde dehydrogenase 2 (ALDH2) and the key DNA base excision repair protein XRCC1. Across cancer types, we found that if one of the corresponding genes exhibits a low expression level, the level of the other gene is increased. Surprisingly, we found that low ALDH2 expression levels associated with high XRCC1 expression levels are indicative for a poor overall survival, particularly in lung and liver cancer patients. In addition, we found that Mithramycin A, a XRCC1 expression inhibitor, efficiently kills cancer cells expressing low levels of ALDH2. CONCLUSIONS: Our data suggest that lung and liver cancers require efficient single-strand break repair for their growth in order to benefit from a low aldehyde detoxification metabolism. We also propose that the ratio of XRCC1 and ALDH2 levels may serve as a useful prognostic tool in these cancer types.

Increased mutability and decreased repairability of a three-lesion clustered DNA-damaged site comprised of an AP site and bi-stranded 8-oxoG lesions.

PURPOSE: Ionizing radiation induces DNA damage, some of which are present in clusters, defined as two or more lesions within one to two helical turns of DNA by passage of a single radiation track. These clusters are thought to contribute to the detrimental effects of radiation, in part due to the compromised repair of clustered DNA damaged sites. MATERIALS AND METHODS: The repair of three-lesion cluster present in oligonucleotides were determined in vitro using the hamster cell line CHO-K1 nuclear extract or purified proteins involved in base excision repair. The mutagenic potential of these clusters present in a plasmid was determined using an Escherichia coli reporter assay. RESULTS: We have shown that the repair of an abasic (AP) site within a three-lesion cluster, comprised of an AP site and bi-stranded 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, is retarded compared to that of an isolated AP site in an in vitro base excision repair (BER) assay. Further, the mutation frequency of the clustered damaged site is up to three times greater than that of an isolated 8-oxoG lesion. CONCLUSIONS: As a consequence of enhanced mutagenic potential of clusters, non-double-strand break (DSB) DNA damage may contribute to the detrimental effects of radiation, in addition to the effects of DSB.

Reduced repair capacity of a DNA clustered damage site comprised of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 2-deoxyribonolactone results in an increased mutagenic potential of these lesions.

A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repair is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase ß, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL.

5,6-Dihydrothymine impairs the base excision repair pathway of a closely opposed AP site or single-strand break.

Ionizing radiation can induce clustered DNA damage (two or more lesions formed within one to two helical turns of DNA by passage of a single ionization track). Using oligonucleotide constructs containing clustered DNA lesions at defined positions, evidence is presented demonstrating that a persistent 5,6-dihydrothymine (DHT) lesion reduces the efficiency of rejoining, in mammalian nuclear extracts, of an opposing AP site or SSB when within 5 bp. The efficiency of repair of the SSB is reduced when DHT is present on the opposing strand in both the 3' and 5' orientation; however, the efficiency of the repair of the AP site is reduced only when DHT is present 3' to the AP site. DNA polymerase beta and ligation are particularly impaired by DHT. It was also shown that in the presence of DHT there is a greater dependence on the long-patch base excision repair pathway than when DHT is absent. In addition, immunodepletion of XRCC1 from the nuclear extracts slows down the initial rate of repair of the AP site in both the presence and absence of DHT, but immunodepletion of XRCC1 has no influence on the repair of an SSB.

Induction of DNA strand breaks, base lesions and clustered damage sites in hydrated plasmid DNA films by ultrasoft X rays around the phosphorus K edge.

To characterize the DNA damage induced by K-shell ionization of phosphorus atom in DNA backbone on the level of hydration, the yields of DNA strand breaks and base lesions arising from the interaction of ultrasoft X rays with energies around the phosphorus K edge were determined using dry and fully hydrated pUC18 plasmid DNA samples. Base lesions and bistranded clustered DNA damage sites were revealed by postirradiation treatment with the base excision repair proteins endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of prompt single-strand breaks (SSBs) with dry DNA irradiated at the phosphorus K resonance energy (2153 eV) is about one-third that below the phosphorus K edge (2147 eV). The yields of prompt double-strand breaks (DSBs) were found to be less dependent on the X-ray energy, with the yields being about two times lower when irradiated at 2153 eV. Heat-labile sites were not produced in detectable amounts. The yields of base lesions were dependent on the energy of the X rays, especially when the DNA was fully hydrated. Bistranded clustered DNA damage sites, revealed enzymatically as additional DSBs, were produced in dry as well as in hydrated DNA with all three energies of X rays. The yields of these enzyme-sensitive sites were also lower when irradiated at the phosphorus K resonance energy. On the other hand, the yields of prompt SSBs and enzyme-sensitive sites for the two off-resonance energies were, larger than those determined previously for gamma radiation. The results indicate that the photoelectric effect caused by X rays and dense ionization and excitation events along the tracks of low-energy secondary electrons are more effective at inducing SSBs and enzyme-sensitive sites. The complex types of damage, prompt and enzymatically induced DSBs, are preferentially induced by phosphorus K resonance at 2153 eV rather than simple SSBs and isolated base lesions, particularly in hydrated conditions. It is concluded that not only the phosphorus K resonance and resulting emission of low-energy LMM-Auger electrons ( approximately 120 eV) but also the level of hydration plays an important role in the induction of complex damage in plasmid DNA.

Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic.

Localized clustering of damage is a hallmark of certain DNA-damaging agents, particularly ionizing radiation. The potential for genetic change arising from the effects of clustered damage sites containing combinations of AP sites, 8-oxo-7,8-dihydroguanine (8-oxoG) or 5,6-dihydrothymine is high. To date clusters containing a DNA base lesion that is a strong block to replicative polymerases, have not been explored. Since thymine glycol (Tg) is non-mutagenic but a strong block to replicative polymerases, we have investigated whether clusters containing Tg are highly mutagenic or lead to potentially cytotoxic lesions, when closely opposed to either 8-oxoG or an AP site. Using a bacterial plasmid-based assay and repair assays using cell extracts or purified proteins, we have shown that DNA double-strand breaks (DSBs) arise when Tg is opposite to an AP site, either through attempted base excision repair or at replication. In contrast, 8-oxoG opposite to Tg in a cluster 'protects' against DSB formation but does enhance the mutation frequency at the site of 8-oxoG relative to that at a single 8-oxoG, due to the decisive role of endonucleases in the initial stages of processing Tg/8-oxoG clusters, removing Tg to give an intermediate with an abasic site or single-strand break.

An AP site can protect against the mutagenic potential of 8-oxoG when present within a tandem clustered site in E. coli.

Ionizing radiation induces clustered DNA damaged sites, defined as two or more lesions formed within one or two helical turns of the DNA through passage of a single radiation track. It is now established that clustered DNA damage sites are found in cells and present a challenge to the repair machinery of the cell but to date, most studies have investigated the effects of bi-stranded lesions. A subset of clustered DNA damaged sites exist in which two or more lesions are present in tandem on the same DNA strand. In this study synthetic oligonucleotides containing an AP site 1, 3 or 5 bases 5' or 3' to 8-oxo-7,8-dihydroguanine (8-oxoG) on the same DNA strand were synthesized as a model of a tandem clustered damaged sites. It was found that 8-oxoG retards the incision of the AP site by exonuclease III (Xth) and formamidopyrimidine DNA glycosylase (Fpg). In addition the rejoining of the AP site by xrs5 nuclear extracts is impaired by the presence of 8-oxoG. The mutation frequency arising from 8-oxoG within a tandem clustered site was determined in both wild type and mutant E. coli backgrounds. In wild-type, nth, fpg and mutY null E. coli, the mutation frequency is slightly elevated when an AP site is in tandem to 8-oxoG, compared with when 8-oxoG is present as a single lesion. Interestingly, in the double mutant mutY/fpg null E. coli, the mutation frequency of 8-oxoG is reduced when an AP site is present in tandem compared with when 8-oxoG is present as a single lesion. This study demonstrates that tandem lesions can present a challenge to the repair machinery of the cell.

8-OxoA inhibits the incision of an AP site by the DNA glycosylases Fpg, Nth and the AP endonuclease HAP1.

Ionizing radiation induces clustered DNA damage sites, whereby two or more individual DNA lesions are formed within one or two helical turns of DNA by a single radiation track. A subset of DNA clustered damage sites exist in which the lesions are located in tandem on the same DNA strand. Recent studies have established that two closely opposed lesions impair the repair machinery of the cell, but few studies have investigated the processing of tandem lesions. In this study, synthetic double-stranded oligonucleotides were synthesized to contain 8-oxoA and an AP site in tandem, separated by up to four bases in either a 5' or 3' orientation. The influence 8-oxoA has on the incision of the AP site by the E. coli glycosylases Fpg and Nth protein and the human AP endonuclease HAP1 was assessed. 8-OxoA has little or no effect on the efficiency of incision of the AP site by Nth protein; however, the efficiency of incision of the AP site by Fpg protein is reduced in the presence of 8-oxoA even up to a four-base separation in both the 5' and 3' orientations. 8-OxoA influences the efficiency of HAP1 incision of the AP site only when it is 3' to the AP site and separated by up to two bases. This study demonstrates that the initial stages of base excision repair can be impaired by the presence of a second base lesion in proximity to an AP site on the same DNA strand. This impairment could have biological consequences, such as mutation induction, if the AP site is present at replication.

Efficiency of repair of an abasic site within DNA clustered damage sites by mammalian cell nuclear extracts.

Ionizing radiation induces clustered DNA damage sites which have been shown to challenge the repair mechanism(s) of the cell. Evidence demonstrating that base excision repair is compromised during the repair of an abasic (AP) site present within a clustered damage site is presented. Simple bistranded clustered damage sites, comprised of either an AP-site and 8-oxoG or two AP-sites, one or five bases 3' or 5' to each other, were synthesized in oligonucleotides, and repair was carried out in xrs5 nuclear extracts. The rate of repair of an AP-site when present opposite 8-oxoG is reduced by up to 2-fold relative to that when an AP-site is present as an isolated lesion. The mechanism of repair of the AP-site shows asymmetry, depending on its position relative to 8-oxoG on the opposite strand. The AP-site is rejoined by short-patch base excision repair when the lesions are 5' to each other, whereas when the lesions are 3' to one another, rejoining of the AP-site occurs by both long-patch and short-patch repair processes. The major stalling of repair occurs at the DNA ligase step. 8-OxoG and an AP-site present within a cluster are processed sequentially, limiting the formation of double-strand breaks to <4%. In contrast, when two AP-sites are contained within the clustered DNA damage site, both AP-sites are incised simultaneously, giving rise to double-strand breaks. This study provides new insight into understanding the processes that lead to the biological consequences of radiation-induced DNA damage and ultimately tumorigenesis.

8-OxoG retards the activity of the ligase III/XRCC1 complex during the repair of a single-strand break, when present within a clustered DNA damage site.

Ionising radiation produces clustered DNA damage. Recent studies have established that the efficiency of excision of a lesion within clustered damage sites is reduced. This study presents evidence that the repair of clustered DNA damage is compromised, relative to that of the isolated lesions, since the lifetime of both lesions is extended by up to eight fold. Simple clustered damage sites, comprised of a single-strand break, one or five bases 3' or 5' to 8-oxoG on the opposite strand, were synthesised in oligonucleotides and repair carried out in XRS5 nuclear extracts. The rate of repair of the single-strand break within these clustered damage sites is reduced, mainly due to inhibition of the DNA ligase III/XRCC1 complex. The single-strand break, present as an isolated lesion, is repaired by short-patch base excision repair, however the mechanism of repair of the single-strand break within the clustered damage site is asymmetric. When the lesions are 5' to each other, the single-strand break is rejoined by short-patch repair whereas the rejoining of the single-strand break occurs by long-patch type repair when the lesions are 3' to one another. The retardation of DNA ligase III/XRCC1 complex, following addition of one base, is responsible for the initiation of long-patch base excision repair when the lesions are 3' to each other. The lesions within the cluster are processed sequentially, the single-strand break being repaired before excision of 8-oxoG, limiting the formation of double-strand breaks to <2%. Stalled processing of clustered DNA damage is suggested to have implications for mutation induction by radiation.

Effect of hydration on the induction of strand breaks and base lesions in plasmid DNA films by gamma-radiation.

The yields of gamma-radiation-induced single- and double-strand breaks (ssb's and dsb's) as well as base lesions, which are converted into detectable ssb by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), at 278 K have been measured as a function of the level of hydration of closed-circular plasmid DNA (pUC18) films. The yields of ssb and dsb increase slightly on increasing the level of hydration (Gamma) from vacuum-dried DNA up to DNA containing 15 mol of water per mole of nucleotide. At higher levels of hydration (15 < Gamma < 35), the yields are constant, indicating that H2O*+ or diffusible hydroxyl radicals, if produced in the hydrated layer, do not contribute significantly to the induction of strand breaks. In contrast, the yields of base lesions, recognized by Nth and Fpg, increase with increasing hydration of the DNA over the range studied. The maximum ratios of the yields of base lesions to that of ssb are 1.7:1 and 1.4:1 for Nth- and Fpg-sensitive sites, respectively. The yields of additional dsb, revealed after enzymatic treatment, increase with increasing level of hydration of DNA. The maximum yield of these enzymatically induced dsb is almost the same as that for prompt, radiation-induced dsb's, indicating that certain types of enzymatically revealed, clustered DNA damage, e.g., two or more lesions closely located, one on each DNA strand, are induced in hydrated DNA by radiation. It is proposed that direct energy deposition in the hydration layer of DNA produces H2O*+ and an electron, which react with DNA to produce mainly base lesions but not ssb. The nucleobases are oxidized by H2O*+ in competition with its conversion to hydroxyl radicals, which if formed do not produce ssb's, presumably due to their scavenging by Tris present in the samples. This pathway plays an important role in the induction of base lesions and clustered DNA damage by direct energy deposition in hydrated DNA and is important in understanding the processes that lead to radiation degradation of DNA in cells or biological samples.

Efficiency of incision of an AP site within clustered DNA damage by the major human AP endonuclease.

A major DNA lesion induced by ionizing radiation and formed on removal of oxidized base lesions by various glycosylases is an apurinic/apyrimidinic site (AP site). The presence of an AP site within clustered DNA damage, induced following exposure to ionizing radiation or radiomimetic anticancer agents, may present a challenge to the repair machinery of the cell, if the major human AP endonuclease, HAP1, does not efficiently incise the AP site. In this study, specific oligonucleotide constructs containing an AP site located at several positions opposite to another damage [5,6-dihydrothymine (DHT), 8-oxoG, AP site, or various types of single strand breaks] on the complementary strand were used to determine the relative efficiency of the purified HAP1 protein in incising an AP site(s) from clustered DNA damage. A base damage (DHT and 8-oxoG) on the opposite strand has little or no influence on the rate of incision of an AP site by HAP1. In contrast, the presence of either a second AP site or various types of single strand breaks, when located one or three bases 3' to the base opposite to the AP site, has a strong inhibitory effect on the efficiency of incision of an AP site. The efficiency of binding of HAP1 to an AP site is reduced by approximately 1 order of magnitude if a single strand break (SSB) is located one or three bases 3' to the site opposite to the AP site on the complementary strand. If the AP site and either a SSB or a second AP site are located at any of the other positions relative to each other, a double strand break may result.