The Structure of the Human AGT Protein Bound to DNA and its Implications for Damage Detection

Interactions of Human O6-Alkylguanine-DNA Alkyltransferase (AGT) with Short Single-stranded DNAs

“…The cooperative binding mechanism described here for single-stranded DNA and reported previously for duplex DNA (13,14) contrasts with the independent binding shown in currently available crystal structures 7 of AGT-DNA complexes (22,48). At present we cannot account for this difference.…”

“…Thus, although the uniformity of packing may decay with increasing template length, the dominant picture remains one of a protein array in which the average separation between the start of two adjacent binding sites is ϳ4 nt. This spacing is significantly shorter than the ϳ8 bp/protein occupied in crystalline AGT complexes formed with duplex DNA (22,48) and the opti- Fig. 5A are Scatchard plots of data obtained from serial dilution and direct titration experiments.…”

“…Because a single molecule of AGT occupies ϳ8 bp of DNA duplex (22,48), it seems likely that AGT molecules overlap along the DNA contour 13 as shown schematically in Fig. 6.…”

“…Torsional stress exerted on duplex DNA may influence the extrusion of alkylguanines from the base stack to form the extrahelical conformation seen in crystalline repair complexes (22,48). Such effects would not be available on single-stranded DNA and may account for the more rapid repair of duplex DNAs than single-stranded DNAs in vitro (19).…”

Direct Reversal of DNA Alkylation Damage

“…The recent crystal structure of hAGT bound to DNA containing a modified cytosine indicates that hydrogen bond donating groups substituted on the para-position of the benzene ring may aid binding of the modified base to the protein. 109 Heterocyclic alkyl groups attached at the O 6 -position of guanine were developed and tested as well. One compound, O 6 -(4-bromoethenyl)guanine (PaTrin 2), had been tested in a phase I clinical trial in combination with the methylating agent temozolomide with positive results.…”