Psoralen interstrand cross-link repair is specifically altered by an adjacent triple-stranded structure

Targeting DNA-damaging agents to specific DNA sites by using sequence-specific DNA ligands has been successful in directing genomic modifications. The understanding of repair processing of such targeted damage and the influence of the adjacent complex is largely unknown. In this way, directed interstrand cross-links (ICLs) have already been generated by psoralen targeting. The mechanisms responsible for ICL removal are far from being understood in mammalian cells, with the proposed involvement of both mutagenic and recombinogenic pathways. Here, a unique ICL was introduced at a selected site by photoactivation of a psoralen moiety with the use of psoralen conjugates of triplex-forming oligonucleotides. The processing of psoralen ICL was evaluated in vitro and in cells for two types of cross-linked substrates, either containing a psoralen ICL alone or with an adjacent triple-stranded structure. We show that the presence of a neighbouring triplex structure interferes with different stages of psoralen ICL processing: (i) the ICL-induced DNA repair synthesis in HeLa cell extracts is inhibited by the triplex structure, as measured by the efficiency of ‘true’ and futile repair synthesis, stopping at the ICL site; (ii) in HeLa cells, the ICL removal via a nucleotide excision repair (NER) pathway is delayed in the presence of a neighbouring triplex; and (iii) the binding to ICL of recombinant xeroderma pigmentosum A protein, which is involved in pre-incision recruitment of NER factors is impaired by the presence of the third DNA strand. These data characterize triplex-induced modulation of ICL repair pathways at specific steps, which might have implications for the controlled induction of targeted genomic modifications and for the associated cellular responses

SAXS Conformational Tracking of Amylose Synthesized by Amylosucrases

Amylose, a linear polymer of α­(1,4)-linked glucosyl units and a major constituent of starch granules, can also be enzymatically synthesized in vitro from sucrose by bacterial amylosucrases. Depending on the initial sucrose concentration and the enzyme used, amylose oligomers (or polymers) are formed and self-associate during synthesis into various semicrystalline morphologies. This work describes for the first time a synchrotron SAXS study of the structure in solution of two amylosucrases, namely, NpAS and the thermostable DgAS, under conditions of polymer synthesis and, simultaneously, the amylose conformation. The structure in solution of both amylosucrases during the reaction was shown to be similar to the known crystallographic structures. The conformation of amylose produced at an early stage consists of a mixture of wormlike chains and double helical cylindrical structures. In the case of NpAS, in a second stage, individual double helices pack into clusters before crystallizing and precipitating. Amylose produced by DgAS never self-associates in such clusters due to the higher temperature used for amylose synthesis. All the dimensions determined for wormlike chains and cylindrical conformations at different times of NpAS synthesis are in very good agreement with structural features usually observed on gels of amylose extracted from starch. This provides new insights in understanding the mechanisms of amylose gelation

Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine PNA conjugated to the topoisomerase I inhibitor camptothecin was found to increase the frequency of repair domain mediated gene correctional events of the EGFP reporter in an in vitro HeLa cell nuclear extract assay, whereas PNA psoralen or chlorambucil conjugates both of which form covalent and also interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression. Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair in nuclear extract. Thus the effects and use of camptothecin conjugates in gene targeted repair merit further studies