Binding of adenosine diphosphoribosyltransferase to the termini and internal regions of linear DNAs.
Binding mechanisms of ADPR-transferase to restricted double-stranded DNA fragments of SV40 and pBR322 DNA were determined by nuclease protection techniques. Top and bottom strands of double-stranded DNA were identified by specific labeling with 32P. Protection against specific exonucleases identified binding of ADPR-transferase to DNA termini, whereas binding to internal regions of linear DNAs was probed by protection against endonucleases. ADPR-transferase protein protected against exonucleolytic attack from lambda exo and exoIII in all DNA fragments tested, demonstrating that the enzyme protein binds indiscriminately to all DNA termini. Extending earlier results [Sastry, S.S., & Kun, E. (1988) J. Biol. Chem. 263, 1505-1512], the modifying effect of the binding of ADPR-transferase to DNA induced changes in DNA conformation, as evident from altered pause sites that appeared following digestion of DNA fragments by lambda exonuclease in the presence of ADPR-transferase. In contrast to the nonselective binding of ADPR-transferase to DNA termini, ADPR-transferase conferred protection endonuclease attack (DNase I and micrococcal nuclease) only to the 209-bp EcoRI-PstI SV40 DNA fragment. These results indicate that binding of ADPR-transferase to relatively rare internal regions of restricted DNA fragments exhibits some degree of specificity. Specificity of binding appears to be related to the coincidental relative A+T-rich regions in DNA, and to DNA bending, both identified in the 209-bp SV40 DNA fragment. Synthetic polydeoxyribonucleotides containing dA-dT bind ADPR-transferase stronger than polydeoxyribonucleotides containing dG-dC. It was deduced from endonuclease protection patterns that binding of the enzyme protein leaves no defined footprints on the 209-bp SV40 DNA fragment, but there is significant modification of DNA structure following binding of the enzyme protein. Methylation protection assays and the prevention of the binding of ADPR-transferase to T4 DNA by its glucosylation indicate that the enzyme binds in the major groove of DNA. The 36-kDa A peptide fragment of ADPR-transferase [Buki, K. G., & Kun, E. (1988) Biochemistry 27, 5990-5995] exhibits the same protection against endonucleolytic enzymes as the intact ADPR-transferase molecule.
Sastry
S S
SS
Department of Pharmacology, University of California, San Francisco 94143.
Buki
K G
KG
Kun
E
E
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology
D004279:DNA, Viral; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D003850:Deoxyribonuclease I