Refined Solution Structure of a DNA Heteroduplex Containing an Aldehydic Abasic Site

Goljer, Igor; Kumar, Satya; Bolton, Philip H.

doi:10.1074/jbc.270.39.22980

Cited by 82 publications

(104 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…NMR Procedures-NMR data were acquired at 500 MHz on a Varian Inova and at 400 MHz on a Varian Unityplus spectrometer, at Wesleyan, and at 500 MHz on a Bruker 500 MHz DMX spectrometer at the University of Wisconsin, Madison, using methods described previously (11,(57)(58)(59) and modified as discussed below. All 31 P experiments were obtained at 161 MHz using the 400-MHz Varian Unityplus.…”

Section: Methodsmentioning

confidence: 99%

“…Quantitation and Assignment of NOE Cross-peaks-The spectra were assigned using the procedures described previously for abasic site-containing DNAs (11,58,62). The standard sequential connectivities of B-form DNA could be used except at the site of the damage.…”

Section: Structures Of Ap Sites In Dnamentioning

confidence: 99%

“…The NOE cross-peak volumes for each of these structures were backcalculated as described previously (11,(57)(58)(59)63) separately using an overall correlation time of 5 ns, a leakage rate of 0.33 s…”

Section: Structures Of Ap Sites In Dnamentioning

confidence: 99%

“…Structure Determination Procedures-Structure refinement by restrained molecular dynamics using X-PLOR 3.1 was performed as described previously (11,(57)(58)(59)63) with the following modifications. The optimum weighting of the NOE constraints was found to be 60 kcal/mol, and the experimental volumes were used as the constraints.…”

Section: Structures Of Ap Sites In Dnamentioning

confidence: 99%

See 3 more Smart Citations

Structures of Apurinic and Apyrimidinic Sites in Duplex DNAs

Beger

Bolton

1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Natural and exogenous processes can give rise to abasic sites with either a purine or pyrimidine as the base on the opposing strand. The solution state structures of the apyrimidinic DNA duplex, with D 6 indicating an abasic site, referred to as AD, and the apurinic DNA duplex with a dC 17 , referred to as CD, have been determined. A particularly striking difference is that the abasic site in CD is predominantly a ␤ hemiacetal, whereas in AD the ␣ and ␤ forms are equally present. Hydrogen bonding with water by the abasic site and the base on the opposite strand appears to play a large role in determining the structure near the damaged site. Comparison of these structures with that of a duplex DNA containing a thymine glycol at the same position as the abasic site and with that of a duplex DNA containing an abasic site in the middle of a curved DNA sequence offers some insight into the common and distinct structural features of damaged DNA sites.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Structures Of Ap Sites In Dnamentioning

confidence: 99%

Section: Structures Of Ap Sites In Dnamentioning

confidence: 99%

Section: Structures Of Ap Sites In Dnamentioning

confidence: 99%

See 2 more Smart Citations

Structures of Apurinic and Apyrimidinic Sites in Duplex DNAs

Beger

Bolton

1998

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Even in the absence of enzyme, the presence of an abasic site lowers the melting temperature (20,22) and confers conformational changes to the adjacent and opposing base pairs (20,21). An abasic site causes perturbations in the helix for one-half a helical turn on the strand opposite the abasic site in the 5Ј direction (23) as shown by accessibility to nucleases S1 and P1.…”

Section: Discussionmentioning

confidence: 99%

Domain Mapping of Human Apurinic/Apyrimidinic Endonuclease

Holt

1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

We recently described the pre-steady state enzymatic binding kinetics of apurinic/apyrimidinic endonuclease (AP endo). In this report we describe the domain structure of the enzyme in solution determined by mild protease digestion in the presence and absence of substrate, product, and an efficient competitive inhibitor (HDP). AP endo is a 35.5-kDa protein with a high degree of homology to its prokaryotic counterpart, exonuclease III (Exo III), except for the amino terminus, which is lacking in the prokaryotic enzyme. The entire conserved region plus an additional 20 residues unique to the eukaryotic enzyme was inaccessible to trypsin and V8 protease, indicating that it forms a tight globular structure. In contrast, the amino-terminal 35 residues were readily accessible to all the proteases investigated, leading us to conclude that they associate poorly with the rest of the structure and constitute a highly fluid region. When AP endo was boiled with SDS and cooled prior to the addition of V8 protease, several acidic residues within the globular domain became protease-accessible, indicating rapid renaturation except along the nuclease fold with restoration of globular conformation for the carboxyl two-thirds of the molecule. Of all the proteases tested, only chymotrypsin was able to cleave internal to the globular portion without prior denaturation. Although AP endo cleaved with chymotrypsin retained full enzymatic activity, the activity was lost when the digested peptides were recovered after denaturation by heat and/or boiling in SDS, precipitation, and renaturation or when fragments were recovered from an SDS gel and renatured. Thus, the protein is probably held together strongly by noncovalent interactions that maintain enzymatic function after protease nicking. The three major chymotrypsin cleavage sites, Tyr-144, Leu-179, and Leu-205, became strikingly less accessible to protease digestion in the presence of abasic site-containing DNA. Since the three residues form a spherical triangle on the surface of the molecule on one side of the nuclease fold, there must be multiple means by which DNA containing an abasic site associates with the enzyme. The most likely explanation is that substrate and product, both of which were present during proteolysis, bind differently to the enzyme. Finally, the two cysteine residues thought to be involved in the redox reaction of AP endo with Jun protein were entirely inaccessible to proteolysis even after prolonged exposure of AP endo to reducing agents. Consequently, if AP endo plays a role in the physiological function of Jun, it must undergo major conformational changes in the process. Alternatively, the two cysteines could maintain an appropriate conformation such that other residues participate directly in the redox activity. Apurinic/apyrimidinic endonuclease (AP endo)1 is a critical enzyme in the repair of abasic sites in DNA. These sites can arise through spontaneous loss of a nucleobase, through the action of N-glycosyl hydrolases, or through oxidative damage of DNA (...

show abstract