Conformational transitions of proteins engaged in DNA double-strand break repair, analysed by tryptophan fluorescence emission and FRET

Abstract

We analysed protein-DNA and protein-protein interactions relevant to the repair of DNA DSBs (double-strand breaks) by NHEJ (non-homologous end-joining). Conformational transitions in mammalian DNA ligases III (LigIII) and IV (LigIV), as well as in PARP-1 [poly(ADP-ribose) polymerase-1], were analysed upon binding to double-stranded DNA by changes in tryptophan emission and FRET (Förster resonance energy transfer) from tryptophan to DNA-conjugated Alexa Fluor® 532. For LigIII, two non-equivalent high- and low-affinity DNA-binding sites are detected interacting sequentially with DNA. PARP-1 displays a single high-affinity DNA-binding site and can displace bound DNA fragments from the low-affinity site of LigIII, consistent with its mediator role in LigIII-DNA interactions. For the LX [LigIV-XRCC4 (X-ray cross-complementation group 4)] complex, a single DNA-binding site is detected. Binding of Ku to DNA was accompanied by conformational changes in the protein and intermolecular FRET from dansyl chromophores of the labelled Ku to the Alexa Fluor® chromophores of Alexa Fluor® 532-conjugated DNA. The average distance of 5.7 nm calculated from FRET data is consistent with a location of Ku at the very end of the DNA molecule. Binding of LX to Ku-DNA complexes is associated with conformational changes in Ku, translocating the protein further towards the DNA ends. The protein-protein and protein-DNA interactions detected and analysed generate a framework for the characterization of molecular interactions fundamental to the function of NHEJ pathways in higher eukaryotes.

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1

“…In CFTR, 11 of the 23 intrinsic Trp residues are situated in transmembrane domains. However, several Trps in NBD1 (W496, W401) and its connecting ICL4 loop (W1063), as well as in NBD2 (W1274, W1282, W1310, W1316) and its connecting ICL2 loop (W278) are buried within the domains or at the domain interface and eligible to report tertiary unfolding of these cytoplasmic domains [58,80]. Because fluorescence emission is an average of all Trps in different local environments, the detected single transition for CFTR suggests that unfolding of these domains is highly cooperative.…”

Poly(N-vinylcaprolactam), a comprehensive review on a thermoresponsive polymer becoming popular

“…Tryptophan residues can strongly bind to DNA [78]. Two amphiphilic copolymers poly[N-vinylcaprolactam-co-(N-t-Boc-tryptophanamido-N'-methacrylthiourea)] (poly(NVCL-co-TrpAMT)) and poly[N-vinylcaprolactam-co-Nvinylpirrolidone-co-(N-t-Boc-tryptophanamido-N'-methacrylthiourea)] (poly(NVCL-co-NVP-co-TrpAMT) were prepared.…”

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

“…The NTase–OBD binds to duplex DNA and maintains a weak DNA nick joining activity in the absence of the Znf–DBD ( 29 ). Tryptophan quenching studies have revealed two DNA binding activities for LigIII with low and high affinity for duplex DNA ( 31 ) that may reflect the binding activities of the ZnF–DBD and NTase–OBD regions, respectively. Two adjacent DNA binding modules in LigIII could serve to align two DNA molecules for intermolecular ligation.…”