Action of Tyrosyl DNA Phosphodiesterase on 3\u27-Phosphoglycolate Terminated DNA Strand Breaks

Free radical-mediated DNA double strand breaks (DSBs) are induced either directly by ionizing radiation or by certain chemicals like bleomycin. These breaks are terminated by 3\u27-PG (PO4CH2COOˉ) or 3\u27-phosphate groups formed as a result of fragmentation of deoxyribose. To study the nature of repair of these 3\u27-blocked breaks, we constructed substrates mimicking free-radical induced DSBs. Human and yeast tyrosyl DNA-phosphodiesterase (Tdpl) efficiently processed substrates with 3\u27-PGs, in either the presence or absence of magnesium, to give a 3\u27-phosphate. Gel filtration chromatography and western blotting codmed that the putative enzyme in human extracts that efficiently processed PG was indeed tyrosyl DNA-phosphodiesterase. When recombinant hTdpl was purified using HiTrap nickel chelating columns and its PG processing activity compared to that of partially purified native enzyme (from lymphoblastoid whole-cell extracts using Sephacryl S-300 gel filtration columns), we found that the recombinant enzyme had lesser 3\u27-PG removal activity than the partially purified native enzyme. On cloning recombinant FLAG-tagged hTdpl into human expression vectors, we observed that the FLAG epitope tag did not show any evidence of affecting the specificity of the enzyme. Due to the many differences between bacterial and human cells, we cloned recombinant FLAG-tagged hTdpl into U-87 cells (adenovirus infected glioma cell) and this recombinant enzyme showed the same specificity toward PG substrates as when prepared from bacteria. End-processing assays using the NHEJ proteins- Ku, DNA-PK and XRCC4/Ligase IV-alone or in combination showed an inhibition of hTdpl activity on 3\u27- overhangs. In nuclear extracts, hTdp1 association with XRCC1, a single-strand repair protein, showed to increase the PG-processing activity of Tdpl up to 4 times. Whole-cell extracts containing mutant Tdpl derived from patients suffering from spinocerebellar axonal neuropathy (SCAN1) were found to be deficient in PG-processing. Addition of JRLl whole-cell extract (SCAN1 extract containing mutant Tdpl) to purified FLAG-tagged hTdpl showed to decrease the phosphotyrosyl processing and increase the PG-processing of FLAG-tagged hTdpl suggesting that there must be other factors in the extract that affect the enzyme activity. Experiments carried out to check for the presence of Tdpl in mitochondrial extracts obtained from GM1310 normal human fibroblasts as well as in SCANl (JRL) mitochondrial extracts, showed that mitochondrial extracts contained Tdpl at a concentration comparable to whole-cell extracts. Our results also showed that mitochondrial extracts from the SCANl cell-line, JRL3 (containing mutant Tdpl), lacked detectable Tdpl activity suggesting that all PG-processing activity in mitochondria may be attributable to Tdpl

Deficiency in 3′-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)

Tyrosyl-DNA phosphodiesterase (TDP1) is a DNA repair enzyme that removes peptide fragments linked through tyrosine to the 3′ end of DNA, and can also remove 3′-phosphoglycolates (PGs) formed by free radical-mediated DNA cleavage. To assess whether TDP1 is primarily responsible for PG removal during in vitro end joining of DNA double-strand breaks (DSBs), whole-cell extracts were prepared from lymphoblastoid cells derived either from spinocerebellar ataxia with axonal neuropathy (SCAN1) patients, who have an inactivating mutation in the active site of TDP1, or from closely matched normal controls. Whereas extracts from normal cells catalyzed conversion of 3′-PG termini, both on single-strand oligomers and on 3′ overhangs of DSBs, to 3′-phosphate termini, extracts of SCAN1 cells did not process either substrate. Addition of recombinant TDP1 to SCAN1 extracts restored 3′-PG removal, allowing subsequent gap filling on the aligned DSB ends. Two of three SCAN1 lines examined were slightly more radiosensitive than normal cells, but only for fractionated radiation in plateau phase. The results suggest that the TDP1 mutation in SCAN1 abolishes the 3′-PG processing activity of the enzyme, and that there are no other enzymes in cell extracts capable of processing protruding 3′-PG termini. However, the lack of severe radiosensitivity suggests that there must be alternative, TDP1-independent pathways for repair of 3′-PG DSBs

Promoter Sequences Prediction Using Relational Association Rule Mining

In this paper we are approaching, from a computational perspective, the problem of promoter sequences prediction, an important problem within the field of bioinformatics. As the conditions for a DNA sequence to function as a promoter are not known, machine learning based classification models are still developed to approach the problem of promoter identification in the DNA. We are proposing a classification model based on relational association rules mining. Relational association rules are a particular type of association rules and describe numerical orderings between attributes that commonly occur over a data set. Our classifier is based on the discovery of relational association rules for predicting if a DNA sequence contains or not a promoter region. An experimental evaluation of the proposed model and comparison with similar existing approaches is provided. The obtained results show that our classifier overperforms the existing techniques for identifying promoter sequences, confirming the potential of our proposal

MiNT-m: An Autonomous Mobile Wireless Experimentation Platform

Limited fidelity of software-based wireless network simulations has prompted many researchers to build testbeds for developing and evaluating their wireless protocols and mobile applications. Since most testbeds are tailored to the needs of specific research projects, they cannot be easily reused for other research projects that may have different requirements on physical topology, radio channel characteristics or mobility pattern. In this paper, we describe the design, implementation and evaluation of MiNT-m, an experimentation platform devised specifically to support arbitrary experiments for mobile multi-hop wireless network protocols. In addition to inheriting the miniaturization feature from its predecessor MiNT [9], MiNT-m enables flexible testbed reconfiguration on an experiment-by-experimen

Reconstitution of 3′-PG processing and subsequent gap filling in SCAN1 extracts by recombinant TDP1

<p><b>Copyright information:</b></p><p>Taken from "Deficiency in 3′-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)"</p><p>Nucleic Acids Research 2005;33(1):289-297.</p><p>Published online 12 Jan 2005</p><p>PMCID:PMC546157.</p><p>© 2005, the authors © </p> The plasmid substrate was treated with 3 mg/ml SCAN1 whole-cell extract (subject 1635) and analyzed as in . In some cases, 25 ng of recombinant TDP1 and/or 200 ng of XRCC4/DNA ligase IV complex (Trevigen) were added. Some samples contained 5 mM EDTA instead of 0.5 mM MgCl, as indicated

Processing of a 3′-PG 14mer by extracts of lymphoblastoid cells from SCAN1 patients (closed symbols) and from unaffected members (open symbols) of the same family, including TDP1+/– carriers (squares, males; circles, females)

<p><b>Copyright information:</b></p><p>Taken from "Deficiency in 3′-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)"</p><p>Nucleic Acids Research 2005;33(1):289-297.</p><p>Published online 12 Jan 2005</p><p>PMCID:PMC546157.</p><p>© 2005, the authors © </p> The substrate was treated with 0.3, 1 or 3 mg/ml of each whole-cell extract (), or with 0.14, 1.4 or 5 mg/ml of each nuclear extract () for 2 h in the presence of EDTA. Following heat denaturation of cellular proteins, the substrate was treated with PNKP to convert 3′-phosphate to 3′-OH termini. Titration with various concentrations of extract () showed that whole-cell extracts (closed circles) contained about twice as much PG-processing activity as nuclear extracts (open squares)