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PubMed2001

Controlling the efficiency of excision repair.

The early studies are recounted, that led to the discovery of the ubiquitous process of DNA excision repair, followed by a review of the pathways of transcription-coupled repair (TCR) and global genomic nucleotide excision repair (GGR). Repair replication of damaged DNA in UV-irradiated bacteria was discovered through the use of 5-bromouracil to density-label newly synthesized DNA. This assay was then used in human cells to validate the phenomenon of unscheduled DNA synthesis as a measure of excision repair and to elucidate the first example of a DNA repair disorder, xeroderma pigmentosum. Features of the TCR pathway (that is defective in Cockayne syndrome (CS)) include the possibility of "gratuitous TCR" at transcription pause sites in undamaged DNA. The GGR pathway is shown to be controlled through the SOS stress response in E. coli and through the activated product of the p53 tumor suppressor gene in human cells. These regulatory systems particularly affect the efficiency of repair of the predominant UV-induced photoproduct, the cyclobutane pyrimidine dimer, as well as that of chemical carcinogen adducts, such as benzo(a)pyrene diol-epoxide. Rodent cells (typically lacking the p53-controlled GGR pathway) and tumor virus infected human cells (in which p53 function is abrogated) are unable to carry out efficient GGR of some lesions. Therefore, caution should be exercised in the interpretation of results from such systems for risk assessment in genetic toxicology. Many problems in excision repair remain to be solved, including the mechanism of scanning the DNA for lesions and the subcellular localization of the repair factories. Also there are persisting questions regarding the multiple options of repair, recombination, and translesion synthesis when replication forks encounter lesions in the template DNA. That is where the field of DNA excision repair began four decades ago with studies on the recovery of DNA synthesis in UV-irradiated bacteria.

Animals↗

PubMed2001

Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro.

The XPC-HR23B complex, a mammalian factor specifically involved in global genomic nucleotide excision repair (NER) has been shown to bind various forms of damaged DNA and initiate DNA repair in cell-free reactions. To characterize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XPC-HR23B for defined lesions on DNA. Here we show that XPC-HR23B preferentially binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to cholesterol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo-G), O6-methylguanine (O6-Me-G), or a single mismatch. Human whole cell extracts could efficiently excise 6-4PPs and cholesterol in an XPC-HR23B-dependent manner, but not 8-oxo-G, O6-Me-G or mismatches. Thus, there was good correlation between the binding specificity of XPC-HR23B for certain types of lesion and the ability of human cell extracts to excise these lesions, supporting the model that XPC-HR23B initiates global genomic NER. Although, XPC-HR23B does not preferentially bind to CPDs, the excision of CPDs in human whole cell extracts was found to be absolutely dependent on XPC-HR23B, in agreement with the in vivo observation that CPDs are not removed from the global genome in XP-C mutant cells. These results suggest that, in addition to the excision repair pathway initiated by XPC-HR23B, there exists another sub-pathway for the global genomic NER that still requires XPC-HR23B but is not initiated by XPC-HR23B. Possible mechanisms will be discussed.

DNA Damage↗

PubMed1998

A new UV-sensitive mutant that suggests a second excision repair pathway in Neurospora crassa.

In an attempt to understand the relationship between photorepair and dark repair in Neurospora crassa, a new mutant was isolated, which showed defects in both repair processes. The new mutant, mus-38, is moderately sensitive to UV and shows imperfect photoreactivation following UV irradiation. DNA was purified from this mutant and the other UV-sensitive mutants, and analyzed for the removal of cyclobutane pyrimidine dimers (CPDs). UV-specific endonuclease-sensitive sites (ESS) completely disappeared with 1 h of photoreactivation in mus-38 DNA, although the survival recovery with photoreactivation was greatly reduced in this mutant. This suggests that the insufficient survival recovery with photoreactivation in mus-38 does not result from a failure of photo-reversal of CPDs. Removal of ESS during liquid holding (dark repair) was slower in mus-38 compared to wild type. To test the possibility that this mutant was involved in excision repair, the double mutant was made between mus-38 and mus-18, which encodes a UV-damage-specific endonuclease. CPD excision in the mus-18 null mutant was severely affected but not completely inhibited. The double mutant showed a complete loss of the excision activity and was super sensitive to UV. These results indicate that mus-38 participates in an excision pathway that is different from the mus-18 pathway. The mus-38 mutant was sensitive not only to UV but also to some chemical mutagens which make adducts on DNA. Thus, mus-38 is possibly involved in an excision-repair pathway that is related to the Saccharomyces cerevisiae RAD3 pathway.

DNA Repair↗

PubMed1991

Breast conservation, the problem of treating the excision site effectively: physical criteria for the choice of technique used.

This paper examines how physical factors such as the depth of the excision cavity below the skin surface and its distance from the underlying lung may impact upon the choice between the use of an electron field (from a Varian Clinac 1800) or an implant (using a double plane of iridium-192 wires). Data have been derived from a phantom dosimetry experiment simulating different permutations of breast size and depth of excision cavity. An anthropomorphic female phantom with two different-sized wax breast phantoms has been used to simulate the clinical circumstances envisaged and isodose distributions have been estimated from an array of TLD readings. The biological significance of doses measured in skin and lung have been examined using the linear-quadratic (LQ) model. Both on physical and biological grounds, the results favour the implant under the experimental conditions adopted. The use of the electron beam to definitively treat the excision cavity (omitting breast tangents) to 60 Gy would result in unacceptable late effects in the skin and an observable incidence of pneumonitis if the excision cavity were near the chest wall. Small carcinogenic risks, particularly to the lung, are apparent with each modality, but may be moderated for the iridium-192 implant by a reduced carcinogenic potential associated with low-dose rate radiation. The use of the newer radionuclides iodine-125 and samarium-145, with less penetrating gamma ray emissions, might be preferred to iridium-192 from the point of view of bronchial carcinogenesis if definitive treatment of the excision cavity became widespread practice.

Brachytherapy↗

PubMed1996

Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily.

BACKGROUND: Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins. RESULTS: Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G. CONCLUSIONS: S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.

Amino Acid Sequence↗

PubMed2000

The piggyBac element is capable of precise excision and transposition in cells and embryos of the mosquito, Anopheles gambiae.

The piggyBac transposable element was tested for transposition activity in plasmid-based excision and inter-plasmid transposition assays to determine if this element would function in Anopheles gambiae cells and embryos. In the Mos55 cell line, precise excision of the piggyBac element was observed only in the presence of a helper plasmid. Excision occurred at a rate of 1 event per 1000 donor plasmids screened. Precise excision of the piggyBac element was also observed in injected An. gambiae embryos, but at a lower rate of 1 excision per 5000 donor plasmids. Transposition of the marked piggyBac element into a target plasmid occurred in An. gambiae cells at a rate of 1 transposition event per 24,000 donor plasmids. The piggyBac element transposed in a precise manner, with the TTAA target site being duplicated upon insertion, in 56% of transpositions observed, and only in the presence of the piggyBac helper. The remaining transpositions resulted in a deletion of target sequence, a novel observation for the phenomenon of piggyBac element insertion. 'Hot spots' for insertion into the target plasmid were observed, with 25 of 34 events involving one particular site. These results are the first demonstration of the precise mobility of piggyBac in this malaria vector and suggest that the lepidopteran piggyBac transposon is a candidate element for germline transformation of anopheline mosquitoes.

Animals↗

PubMed2002

Strategies in the management of mid and distal rectal cancer with total mesorectal excision.

In the last two decades, dramatic improvement in outcome has been made in the management of rectal cancer. This has been brought about mainly by advancements in surgical technique for radical resection. With the recognition of the importance of the circumferential margin and presence of spread in the lymphovascular tissues in the mesorectum, total mesorectal excision is now commonly recognized as the optimal surgical technique for cancer of the mid and distal rectum. Not only have local control and disease-specific survival improved with the practice of total mesorectal excision, but various bodily functions have also been preserved following surgery for rectal cancer. New issues have arisen with the practice of total mesorectal excision and the strategies for management of rectal cancer require re-evaluation. In this article, the rationale and the outcomes of total mesorectal excision are reviewed. Issues such as the high anastomotic leakage rate following sphincter-preserving surgery, the poor results of abdominoperineal resection, the role of adjuvant therapy and bowel function disturbances will be addressed. Lastly, the status of the laparoscopic approach to rectal cancer with the principle of total mesorectal excision are discussed.

Humans↗

PubMed1998

Sphincter preservation in rectal cancer. Preoperative radiation therapy followed by local excision.

Radiation therapy followed by local excision results in local control rates that appear comparable to those of local excision alone (in highly selected patients) or local excision followed by adjuvant radiation therapy. A significant drawback of this approach, however, is the potential loss of important histological information, such as risk of lymph node metastasis, depth of tumor penetration, and presence of lymphatic or vascular invasion. Radiation therapy followed by local excision may be an option for treatment of more advanced T3 rectal cancers in patients who either refuse radical surgery or are medically unfit. The available data in the literature do not support the routine use of local excision after radiation therapy in otherwise healthy patients with locally advanced rectal cancer.

Anal Canal↗

PubMedDate not supplied

Proximal clavicle excision: an analysis of results.

Medial clavicle excision has been reported by several authors, but few cases are documented, and long-term follow-up information is lacking. The purpose of this study was to examine the long-term results of medial clavicle excision in regard to function, pain, cosmesis, and complications. Fifteen patients ranging in age from 18 to 64 years (average 43 years) were evaluated an average of 4.6 years (range 1 to 14 years) after proximal clavicle excision. The indications for excision were unstable anterior subluxation/dislocation of the sternoclavicular joint (four cases), unstable posterior dislocation (one case), sternoclavicular osteoarthritis (nine cases), and proximal clavicle osteomyelitis (one case). An average of 2.9 cm of the medial clavicle was excised (range 1 to 4 cm). Fourteen of the 15 patients received significant relief of pain. On a strict grading scale four patients had an excellent result, five a good result, four a fair result, and two a poor result. Regeneration of the clavicle appeared to contribute to a poor result. No operative complications occurred. These findings aid our understanding of surgical options and outcome in the treatment of sternoclavicular joint disease.

Adult↗

PubMed1999

Excision of endometriosis with laparosonic coagulating shears.

Excision of endometriotic lesions allows the diagnosis to be confirmed and may provide long-term relief of symptoms. Laparoscopic excision is generally accepted for treatment of all stages of disease and can be accomplished safely with laser, electrosurgery, or ultrasonic energy. Ultrasonic energy uses the 5-mm hook blade for excision and 5-mm harmonic scalpel ball electrode for coagulation. Disease was excised by laparosonic coagulating shears (LCS) in 14 women. All patients had an uneventful postoperative course, with 13 (93%) remaining pain free 7 to 18 months postoperatively. The LCS is easy to use and allows endometriosis to be excised with an adequate margin of safety. (J Am Assoc Gynecol Laparosc 6(2):195-199, 1999)

Electrocoagulation↗

PubMed2003

Mechanism of 5'-directed excision in human mismatch repair.

We have developed a purified system that supports mismatch-dependent 5'-->3' excision. In the presence of RPA, ATP, and a mismatch, MutSalpha activates 5'-->3' excision by EXOI, and excision terminates after removal of the mispair. MutSalpha confers high processivity on EXOI, and termination is due to RPA-dependent displacement of this processive complex from the helix and a weak ability of EXOI to reload at the RPA-bound gap in the product, as well as MutSalpha- and MutLalpha-dependent suppression of EXOI activity in the absence of a mismatch cofactor. As observed in the purified system, excision directed by a 5' strand break in HeLa nuclear extract can proceed in the absence of MutLalpha or PCNA, although 3' excision in the extract system requires both proteins.

Adenosine Triphosphatases↗

PubMed2002

Opposite base specificity in excision of pyrimidine ring-opened 1,N6-ethenoadenine by thymine glycol-DNA-glycosylases.

A highly mutagenic DNA lesion, 1,N6-ethenoadenine ( epsilon A) is chemically unstable and either depurinates or converts to a pyrimidine ring-opened product upon water molecule addition to the C(2)z.sbnd;N(3) bond in epsilon dA (compound B). Compound B subsequently undergoes deformylation to yield compound C, which depurinates in the final step of the epsilon A rearrangement pathway. We have previously shown that epsilon A rearrangement products are not repaired by human N-methylpurine-DNA-glycosylase, which excises parental epsilon A. Compound B was shown to be eliminated from a B:T pair by Escherichia coli formamidopyrimidine-DNA-glycosylase (Fpg protein) and endonuclease III (Nth protein). Fpg protein excised B also from a B:C pair, and much less efficiently from B:A and B:G pairs [J. Biol. Chem. 276 (2001) 21821]. Here we show that efficiency of B excision by the Nth protein also depends on the opposite base in the pair. Most efficient repair is observed when this derivative is paired with dG (Km=18nM, kcat=12) and is less favourable when paired with dC (Km=40nM, kcat=13) and dT (Km=32nM, kcat=11). In physiological conditions, compound B is probably not excised by the Nth-glycosylase from a B:A pair, or from a single-stranded DNA, since kinetic constants in these conditions are an order or two orders of magnitude higher than when B is paired with T, C or G. A similar specificity for B excision was found for Saccharomyces cerevisiae Ntg2-glycosylase. Thus, when paired with A, an epsilon A derivative might be more persistent than when paired with other bases and give rise to AT-->TA transversions.

Adenosine↗

PubMed2003

Long and short mRnas transcribed from the medaka fish transposon Tol2 respectively exert positive and negative effects on excision.

The medaka fish transposable element, Tol2, is a member of the hAT family of transposons. It has been directly demonstrated to be active and two mRNAs, differing in length, have been isolated. They cover exons 1-4 and exons 2-4 and the longer form has already been proven to catalyse transposition reactions. However, the function of the shorter mRNA in medaka cells has hitherto remained unclear. In the present study, first we constructed a quantitative system to detect Tol2 excision using an indicator plasmid carrying a non-autonomous Tol2 within its lacZ gene; second we injected mRNAs with the plasmid into medaka eggs. Excision of Tol2 was detected as E. coli blue colonies caused by the recovery of lacZ activity. Addition of the longer mRNA increased excision, but the shorter did not. Moreover, co-injection of both mRNAs greatly lowered the frequency compared with the case of treatment with the longer mRNA alone. These results indicate that the shorter mRNA has an inhibitory effect on the excision reaction, and that the N-terminal region of the transposase encoded by exon 1, including a BED zinc finger, presumably plays an important role in excision. Here, we suggest a regulatory mechanism of Tol2 transposition involving the expression of these mRNAs.

Animals↗

PubMed1997

P element excision in Drosophila is stimulated by gamma-irradiation in transient embryonic assays.

The influence of gamma-irradiation on P element excision and excision-site repair mechanisms was directly tested by embryonic somatic excision assays. Preblastoderm P[ry+, delta 2-3](99B) embryos, having a stable source of somatically active P transposase, were irradiated previous to injection with P excision indicator plasmids. Frequencies of precise or nearly precise P excision increased with gamma-ray doses ranging from 0.5 to 3.5 Gy. Higher doses resulted in frequencies close to that in unirradiated embryos, though considerable embryonic lethality was also evident at these doses. A direct positive interaction between gamma-irradiation and P element activity is concluded.

Animals↗

PubMed1991

Tailoring of cortical excisions for frontal lobe epilepsy.

Follow-up data of 2 to 49 years' duration are presented on 257 patients with cortical excisions for non-tumoral frontal lobe epilepsy. Twenty-six percent have become and remained seizure free. Another 30% have had a marked reduction of seizure tendency. Thus a total of 56% have had a long term complete or marked reduction of seizure tendency after the cortical resection. The cortical resections were classified into 5 regional categories. Forty-seven percent of patients with anterior frontal resections were in the seizure free category, as were 35% of patients with excisions limited to the convexity cortex and 32% of patients with maximal or near maximal frontal lobectomy. Only 18% of patients with frontal parasaggital excisions were in the seizure free category, and 10% of the patients who had excision of frontal cortex plus limited excision of adjacent central and/or temporal cortex.

Adolescent↗

PubMed2000

AlkA protein is the third Escherichia coli DNA repair protein excising a ring fragmentation product of thymine.

Various forms of oxidative stress lead to the formation of damaged bases including N-(2-deoxy-beta-D-erythro-pentofuranosyl)-N-3-(2R-hydroxyisobutyric acid)-urea or alphaRT, the fragmentation product of thymine formed from 5R-thymidine C5-hydrate upon hydrolysis. It was shown that alphaRT is excised by Escherichia coli Fpg and Nth proteins. Here we report that when present in DNA, alphaRT is, in addition, a substrate for the E. coli AlkA protein with an apparent K(m) value of congruent with170 nM. alphaRT positioned opposite T, dG, dC, and dA were efficiently excised by AlkA protein from duplex oligodeoxynucleotides in the following order: dA approximately T >> dC approximately dG. This is the first example of the excision of a ring opened form of a pyrimidine by AlkA protein and also the first example where the same DNA base lesion is excised by three different DNA glycosylases of the base excision repair pathway. The present results suggest possible structural similarity of the active site between E. coli AlkA, Fpg, and Nth proteins.

5' Untranslated Regions↗

PubMed1991

Specificities and kinetics of uracil excision from uracil-containing DNA oligomers by Escherichia coli uracil DNA glycosylase.

Uracil DNA glycosylase excises uracil residues from DNA that can arise as a result of deamination of cytosine or incorporation of dUMP residues by DNA polymerase. We have carried out a detailed study to define the specificities and the kinetic parameters for its substrates by using a number of synthetic oligodeoxyribonucleotides of varying lengths and containing uracil residue(s) in various locations. The results show that the Escherichia coli enzyme can remove a 5'-terminal U from an oligomer only if the 5'-end is phosphorylated. The enzyme does not remove U residues from a 3'-terminal position, but U residues can be excised from oligonucleotides with either pd(UN)p or pd(UNN) 3'-termini. The oligomer d(UUUUT) can have the second or third U residues from the 5'-end excised even when the neighboring site is an abasic site (3' or 5', respectively). On the basis of these findings, pd(UN)p was anticipated to be the smallest size substrate. Results show detectable amounts of U release from the substrate pd(UT)p; however, significantly higher amounts of U release were observed from pd(UT-sugar) or pd(UTT). Determinations of the Km and Vmax values show that the different rates of U excision from oligomers of different sizes (trimeric to pentameric) but containing U in the same position are largely due to the differences in the Km values, whereas the different rates of U excision from the substrates of the same size but containing U in different positions are largely due to different Vmax values.

DNA Glycosylases↗

PubMed2001

Recognition and kinetics for excision of a base lesion within clustered DNA damage by the Escherichia coli proteins Fpg and Nth.

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damages that are thought to lead to deleterious biological consequences, due to the challenge that clustered damage may present to the repair machinery of the cell. Specific oligonucleotides, containing either dihydrothymine (DHT) or 7,8-dihydro-8-oxoguanine (8-oxoG) opposite to specific lesions at defined positions on the complementary strand, have been used to determine the kinetic constants, K(M), k(cat), and specificity constants, for excision of DHT and 8-oxoG by the Escherichia coli base excision repair proteins, endonuclease III (Nth) and formamidopyrimidine glycosylase (Fpg), respectively. For excision of DHT opposite to 8-oxoadenine by Nth or Fpg proteins, or 8-oxoG opposite to 8-oxoG by Fpg, the major change in the specificity constant occurs when the second lesion on the complementary strand is one base to the site opposite to DHT or 8-oxoG. The specificity constants for excision of DHT or 8-oxoG by both proteins are reduced by up to 2 orders of magnitude when an abasic site or a strand break is opposite on the complementary strand. Whereas the values of K(M) are only slightly affected by the presence of a second lesion, the major change is seen as a reduction in the values of k(cat). The binding of Fpg protein to oligonucleotides containing 8-oxoG is inhibited, particularly when a single strand break is near to 8-oxoG on the complementary strand. It is inferred that not only the binding affinity of Fpg protein to the base lesion but also the rate of excision of the damaged base is reduced by the presence of another lesion, particularly a single strand break or an AP site on the complementary strand.

Apurinic Acid↗