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Conformational analysis of DNA repair intermediates by time-resolved fluorescence spectroscopy.

ABSTRACT: DNA repair enzymes are essential for maintaining the integrity of the DNA sequence. Unfortunately, very little is known about how these enzymes recognize damaged regions along the helix. Structural analysis of cellular repair enzymes bound to DNA reveals that these enzymes are able to recognize DNA in a variety of conformations. However, the prevalence of these deformations in the absence of enzymes remains unclear, as small populations of DNA conformations are often difficult to detect by NMR and X-ray crystallography. Here, we used time-resolved fluorescence spectroscopy to examine the conformational dynamics of linear, nicked, gapped, and bulged DNA in the absence of protein enzymes. This analysis reveals that damaged DNA is polymorphic in nature and able to adopt multiple individual conformations. We show that DNA repair intermediates that contain a one-nucleotide gap and bulge have a significant propensity to adopt conformations in which the orphan base resides outside the DNA helix, while DNA structures damaged by a nick or two-nucleotide gap favor intrahelical conformations. Because changes in DNA conformation appear to guide the recognition of DNA repair enzymes, we suggest that the current approach could be used to study the mechanism of DNA repair.

SUBMITTER: Lin S

PROVIDER: S-EPMC2733762 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Conformational analysis of DNA repair intermediates by time-resolved fluorescence spectroscopy.

Lin Su S Horning David P DP Szostak Jack W JW Chaput John C JC

The journal of physical chemistry. A 20090901 35

DNA repair enzymes are essential for maintaining the integrity of the DNA sequence. Unfortunately, very little is known about how these enzymes recognize damaged regions along the helix. Structural analysis of cellular repair enzymes bound to DNA reveals that these enzymes are able to recognize DNA in a variety of conformations. However, the prevalence of these deformations in the absence of enzymes remains unclear, as small populations of DNA conformations are often difficult to detect by NMR a ...[more]

PMID: 19673467

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Project description:Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 °C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.

Dataset Information

Conformational analysis of DNA repair intermediates by time-resolved fluorescence spectroscopy.

Publications

Conformational analysis of DNA repair intermediates by time-resolved fluorescence spectroscopy.

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