Polymerase chain reaction (PCR) is one of the most important laboratory techniques used in molecular biology, genetics and molecular diagnostics. The success of a PCR-based method largely depends on the correct nucleic acid sequence analysis in silico prior to a wet-bench experiment. Here, we report the development of an online Java-based software for virtual PCR on linear or circular DNA templates and multiple primer or probe search from large or small databases. Primer or probe sensitivity and specificity are predicted by searching a database to find sequences with an optimal number of mismatches, similarity and stability. The software determines primer location, orientation, efficiency of binding and calculates primer melting temperatures for standard and degenerate oligonucleotides. The software is suitable for batch file processing, which is essential for automation when working with large amounts of data. The online Java software is available for download at http://primerdigital.com/tools/pcr.html. Accession numbers for the sequences resulting from this study: EU140956 EU177767 EU867815 EU882730 FJ975775-FJ975780 HM481419 HM481420 KC686837-KC686839 KM262797.
DNA-dependent poly(ADP-ribose) polymerases (PARPs) PARP1, PARP2 and PARP3 act as DNA break sensors signalling DNA damage. Upon detecting DNA damage, these PARPs use nicotine adenine dinucleotide as a substrate to synthesise a monomer or polymer of ADP-ribose (MAR or PAR, respectively) covalently attached to the acceptor residue of target proteins. Recently, it was demonstrated that PARP1-3 proteins can directly ADP-ribosylate DNA breaks by attaching MAR and PAR moieties to terminal phosphates. Nevertheless, little is still known about the mechanisms governing substrate recognition and specificity of PARP1, which accounts for most of cellular PARylation activity. Here, we characterised PARP1-mediated DNA PARylation of DNA duplexes containing various types of breaks at different positions. The 3′-terminal phosphate residue at doublestrand DNA break ends served as a major acceptor site for PARP1-catalysed PARylation depending on the orientation and distance between DNA strand breaks in a single DNA molecule. A preference for ADP-ribosylation of DNA molecules containing 3′-terminal phosphate over PARP1 auto-ADPribosylation was observed, and a model of DNA modification by PARP1 was proposed. Similar results were obtained with purified recombinant PARP1 and HeLa cell-free extracts. Thus, the biological effects of PARP-mediated ADP-ribosylation may strongly depend on the configuration of complex DNA strand breaks.One of the earliest DNA damage response events in the cell is the recruitment of DNA-dependent poly(ADP-ribose) polymerases 1, 2 and 3 (PARP1-3) to the sites of DNA strand breaks 1-3 . PARPs 1-3 are catalytically activated through interaction with DNA strand discontinuities and catalyse poly(ADP-ribosyl)ation (PARylation) or mono(ADP-ribosyl)ation (MARylation, in case of PARP3) of nuclear acceptor proteins including auto-ADP-ribosylation using NAD + as the ADP-ribose donor 4-6 . Protein ADP-ribosylation provides a scaffold for the recruitment of other proteins, which also become potential targets for PARP-dependent ADP-ribosylation altering the function of the modified proteins and coordinating the choice of DNA break processing and repair pathways. PARP1 is one of the most abundant nuclear proteins and accounts for ~80-90% of the PARylation activity in the cell induced by DNA damage 7,8 . PARP1 is recruited to damage sites in genomic DNA within a few seconds after laser micro-irradiation 3 and modulates multiple pathways involved in DNA strand break repair: base excision repair, nucleotide excision repair, homologous recombination and non-homologous end-joining 4,9 . Depletion of PARP1 results in hypersensitivity to ionising radiation, to oxidative stress, and to alkylating agents 10 .Recently, the previously unknown phenomenon of post-replicative reversible ADP-ribosylation of DNA strand break termini catalysed by mammalian PARP1-3 was uncovered. These PARPs catalyse covalent addition of ADP-ribose units to 5′-and 3′-terminal phosphates and to 2′-OH termini of modified nucleotides at DNA strand breaks, thereb...
Fanconi anemia (FA) is a recessive cancer prone syndrome featuring bone marrow failure and hypersensitivity to DNA interstrand crosslinks (ICLs) and, to a milder extension, to ionizing radiation and oxidative stress. Recently, we reported that human oxidative DNA glycosylase, NEIL1 excises with high efficiency the unhooked crosslinked oligomer within three-stranded DNA repair intermediate induced by photoactivated psoralen exposure. Complete reconstitution of repair of the ICL within a three-stranded DNA structure shows that it is processed in the short-patch base excision repair (BER) pathway. To examine whether the DNA damage hypersensitivity in FA cells follows impaired BER activities, we measured DNA glycosylase and AP endonuclease activities in cell-free extracts from wild-type, FA, and FA-corrected cells. We showed that immortalized lymphoid cells of FA complementation Groups A, C, and D and from control cells from normal donors contain similar BER activities. Intriguingly, the cellular level of NEIL1 protein strongly depends on the intact FA pathway suggesting that the hypersensitivity of FA cells to ICLs may, at least in part, arise from downregulation or degradation of NEIL1. Consistent with this result, plasmid-based expression of the FLAG-tagged NEIL1 protein partially complements the hypersensitivity FA cells to the crosslinking agents exposures, suggesting that NEIL1 specifically complements impaired capability of FA cells to repair ICLs and oxidative DNA damage. These findings shed light to how the FA pathway may regulate DNA repair proteins and bring explanation for the long-time disputed problem of the oxidative stress sensitive phenotype of FA cells.
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