Site-specific DNA alkylation and repair

Thesis/Dissertation · Fri Jan 01 04:00:00 EST 1993

OSTI ID:6965255

Ezaz-Nikpay, K

This thesis describes a general method for the site-specific insertion of modified nucleotides into DNA and the application of this method to the study of N7-methyl-2[prime]-deoxyguanosine (m[sup 7]dG) in DNA. This thesis describes the chemical basis for the gap insertion/ligation method (GIL) and the use of this method to generate circularly permuted oligonucleotides. In this method, the synthesis of a single oligonucleotide leads to the formation of a double-stranded multimer with periodically-occurring gaps upon base-pairing in solution. The sequential action of a DNA polymerase and a DNA ligase leads to the insertion of a 2[prime]-deoxynucleoside-5[prime]-triphosphate into the gap, and formation of covalently-closed DNA. Finally, restriction endonucleases are used to generate oligonucleotides which contain the introduced nucleotide at symmetrically-related positions. The author describes the use of the GIL method for the insertion of m[sup 7]dG into various oligonucleotides and the Dickerson/Drew dodecamer respectively. The Dickerson/Drew dodecamer was chosen because it has been extensively studies both in its native and adduct bearing forms. The author describes the biophysical characterization of m[sup 7]dG in DNA, and concludes that the probe moiety in dimethyl-sulfate and template-directed interference footprinting of protein-DNA complexes in m[sup 7]dG and not a product of its decomposition. Further studies of m[sub 7]dG in DNA reveal that over long periods of time, the primary product of decomposition is an apurinic site. This dissertation describes the large-scale synthesis of the Dickerson/Drew dodecamer, and the characterization of its effect on DNA structure using nuclear magnetic resonance spectroscopy. The final chapter describes the overproduction, purification and crystallization of N3-methyladenine DNA glycosylase II (AlkA). AlkA is known to repair m[sup 7]dG residues in DNA.

• Research Organization: Harvard Univ., Cambridge, MA (United States)
• OSTI ID: 6965255
• Country of Publication: United States
• Language: English

Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
400201 -- Chemical & Physicochemical Properties
550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
ADDUCTS
AROMATICS
AZAARENES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CHEMICAL REACTIONS
DNA
DNA ADDUCTS
DNA REPAIR
GUANOSINE
HETEROCYCLIC COMPOUNDS
KINETICS
METHYLATION
MOLECULAR BIOLOGY
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PURINES
REACTION KINETICS
REPAIR
RIBOSIDES