Mechanisms for the processing of a frozen topoisomerase-DNA conjugate by human cell-free extracts.
The metabolic fate of covalently linked DNA-protein complexes (cross-links) is not clearly understood. Our aim was to investigate the processing of protein-DNA cross-links by cellular enzymes. As an example of a DNA-protein cross-link, we have constructed frozen topoisomerase-DNA conjugates and investigated their processing by human cell-free extracts. A suicide DNA substrate was constructed that upon reaction with vaccinia type I topoisomerase yielded a highly stable covalent DNA-protein cross-link. When this conjugate was treated with human nuclear or whole cell extracts, two sites of DNA breakpoints were detected: one set of double-stranded breaks occurred close to the 3' side of the topoisomerase (topo) conjugation site, and there was another set of nicks about 30 nucleotides 3' to the topo site. The double-stranded breaks were not made by extracts from xeroderma pigmentosum group A mutant cells, suggesting that the xeroderma pigmentosum group A damage recognition protein may be required for the occurrence of DNA breakage. In addition to these DNA breakage reactions, there was an activity that resulted in the delinking of the frozen topoisomerase (or proteolytic fragments thereof) from the DNA substrate, which was followed by a ligation step that restored the continuity of the broken DNA strand at the erstwhile topo attachment site. We suggest that frozen topoisomerase-DNA conjugates (and perhaps other types of covalent DNA-protein complexes) are processed by multiple pathways that may involve the cleavage of the DNA in the covalent protein-DNA complex and/or enzymatic delinking followed by ligation of the broken DNA ends. These processes may represent the "repair" of DNA-protein cross-links.
Sastry
S
S
Laboratory of Molecular Genetics, The Rockefeller University, New York, New York 10021, USA. sastrys@rockvax.rockefeller.edu
Ross
B M
BM
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002474:Cell-Free System; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D014983:Xeroderma Pigmentosum
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004264:DNA Topoisomerases, Type I