Protein-DNA footprinting by endcapped duplex oligodeoxyribonucleotides

Ng, Pei-Sze

doi:10.1093/nar/gnh103

Cited by 14 publications

(12 citation statements)

References 31 publications

(32 reference statements)

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“…This enhanced stabilization could be useful for increasing the pre-organization of DNA duplex fragments that are commonly involved in the recognition and binding of enzymes involved in degradation, repair, and synthesis of nucleic acids. We have previously shown that DNA ‘endcapped’ at both ends by non-nucleotide linkers are useful for stabilizing short duplex sequences for studying the DNA footprint of T4 DNA ligase (11). …”

Section: Introductionmentioning

confidence: 99%

Synthesis and Evaluation of New Spacers for Use as dsDNA End-Caps

Laing

Balasundarum

et al. 2010

Bioconjugate Chem.

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A series of aliphatic and aromatic spacer molecules designed to cap the ends of DNA duplexes have been synthesized. The spacers were converted into dimethoxytrityl protected phosphoramidites as synthons for oligonucleotides synthesis. The effect of the spacers on the stability of short DNA duplexes was assessed by melting temperature studies. Endcaps containing amide groups were found to be less stabilizing than the hexaethylene glycol spacer. Endcaps containing either a terthiophene or a naphthalene tetracarboxylic acid dimide were found to be significantly more stabilizing. The former showed a preference for stacking above an A•T base pair. Spacers containing only methylene (-CH 2 -) and amide (-CONH-) groups interact weakly with DNA and consequently may be optimal for applications that require minimal influence on DNA structure but require a way to hold the ends of double-stranded DNA together.

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Section: Introductionmentioning

confidence: 99%

Synthesis and Evaluation of New Spacers for Use as dsDNA End-Caps

Laing

Balasundarum

et al. 2010

Bioconjugate Chem.

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“…The great decrease in ligation activity was thus not the result of the influence of another azobenzene moiety, which is six nucleotides away. [19] The difference might be the result of the different bases adjacent to the azobenzene moiety (i.e., depending strongly on the adjacent sequence; vide infra).…”

Section: Resultsmentioning

confidence: 99%

“…According to Ng et al, five base pairs on the 5'-side and six base pairs at the 3'-side of the ligation site are necessary for ligation by T4 DNA ligase. [19] It can be deduced that T4 DNA ligase has to cover at least these 11 base pairs in total for ligation. Here, we changed the position of the ligation site to investigate the effect of an introduced azobenzene on DNA ligation ( Figure 6).…”

Section: Wwwchemeurjorgmentioning

confidence: 99%

Nick Sealing by T4 DNA Ligase on a Modified DNA Template: Tethering a Functional Molecule on D‐Threoninol

Liang

Fujioka

Asanuma

2011

Chemistry A European J

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Efficient DNA nick sealing catalyzed by T4 DNA ligase was carried out on a modified DNA template in which an intercalator such as azobenzene had been introduced. The intercalator was attached to a D-threoninol linker inserted into the DNA backbone. Although the structure of the template at the point of ligation was completely different from that of native DNA, two ODNs could be connected with yields higher than 90% in most cases. A systematic study of sequence dependence demonstrated that the ligation efficiency varied greatly with the base pairs adjacent to the azobenzene moiety. Interestingly, when the introduced azobenzene was photoisomerized to the cis form on subjection to UV light (320-380 nm), the rates of ligation were greatly accelerated for all sequences investigated. These unexpected ligations might provide a new approach for the introduction of functional molecules into long DNA strands in cases in which direct PCR cannot be used because of blockage of DNA synthesis by the introduced functional molecule. The biological significance of this unexpected enzymatic action is also discussed on the basis of kinetic analysis.

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“…In many cases, it could be advantageous to use non-nucleotide connectors to stabilize the end without introducing additional charge. For example, polyethylene glycol connects are available for commercial DNA synthesis, and these have been shown to effectively stabilize oligonucleotide ends (40,41). We did not use such hairpin structures in the current study because APE1 has robust endonuclease activity towards this type of linkage (14).…”

Section: Discussionmentioning

confidence: 99%

Defining the functional footprint for recognition and repair of deaminated DNA

Baldwin

O’Brien

2012

Nucleic Acids Research

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Spontaneous deamination of DNA is mutagenic, if it is not repaired by the base excision repair (BER) pathway. Crystallographic data suggest that each BER enzyme has a compact DNA binding site. However, these structures lack information about poorly ordered termini, and the energetic contributions of specific protein–DNA contacts cannot be inferred. Furthermore, these structures do not reveal how DNA repair intermediates are passed between enzyme active sites. We used a functional footprinting approach to define the binding sites of the first two enzymes of the human BER pathway for the repair of deaminated purines, alkyladenine DNA glycosylase (AAG) and AP endonuclease (APE1). Although the functional footprint for full-length AAG is explained by crystal structures of truncated AAG, the footprint for full-length APE1 indicates a much larger binding site than is observed in crystal structures. AAG turnover is stimulated in the presence of APE1, indicating rapid exchange of AAG and APE1 at the abasic site produced by the AAG reaction. The coordinated reaction does not require an extended footprint, suggesting that each enzyme engages the site independently. Functional footprinting provides unique information relative to traditional footprinting approaches and is generally applicable to any DNA modifying enzyme or system of enzymes.

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Protein-DNA footprinting by endcapped duplex oligodeoxyribonucleotides

Cited by 14 publications

References 31 publications

Synthesis and Evaluation of New Spacers for Use as dsDNA End-Caps

Synthesis and Evaluation of New Spacers for Use as dsDNA End-Caps

Nick Sealing by T4 DNA Ligase on a Modified DNA Template: Tethering a Functional Molecule on D‐Threoninol

Defining the functional footprint for recognition and repair of deaminated DNA

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