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Replicative helicases can translocate through abasic site-induced covalent topoisomerase IV-DNA complexes
1Department of Pharmacology, University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA.
Abstract:
Some topoisomerase inhibitors trap covalent topoisomerase-DNA complexes as topoisomerase-drug-DNA ternary complexes. Ternary complex formation results in inhibition of DNA replication and generation of permanent double-strand breaks. Recent demonstrations of the stimulation of covalent topoisomerase-DNA complex formation by DNA lesions suggest that DNA damage may act as an endogenous topoisomerase poison. We have investigated the effects of abasic (AP) sites on topoisomerase IV (Topo IV). AP sites can stimulate the formation of covalent Topo IV-DNA complexes when they are located either within the 4 base overhang generated by DNA scission or immediately 5' to the point of scission (the -1 position). Thus, the AP site acts as a position-specific, endogenous topoisomerase poison. Both EDTA and salt can reverse covalent Topo IV-DNA complexes induced by AP sites located within the 4 base overhang. Interestingly, an AP site at the -1 position inhibits EDTA-mediated reversal of formation of the covalent Topo IV-DNA complex. Furthermore, we find that, unlike quinolone-induced covalent Topo IV-DNA complexes, AP site-induced covalent Topo IV-DNA complexes do not inhibit the helicase activities of the DnaB and T7 Gene 4 proteins. These results suggest that the AP site-induced poisoning of Topo IV does not arrest replication fork progression.
Insights
DNA damage, specifically abasic (AP) sites, can act as endogenous poisons for topoisomerase IV (Topo IV). AP sites at specific positions can trap Topo IV-DNA complexes, influencing DNA replication fork progression.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Topoisomerase inhibitors are crucial in cancer therapy, forming ternary complexes that halt DNA replication and induce breaks.
- DNA lesions can potentially act as endogenous topoisomerase poisons, mimicking drug effects.
- Topoisomerase IV (Topo IV) plays a vital role in DNA decatenation and replication.
Purpose of the Study:
- To investigate the impact of abasic (AP) sites on Topoisomerase IV (Topo IV) activity.
- To determine if AP sites can function as endogenous topoisomerase poisons.
- To elucidate the consequences of AP site-induced Topo IV-DNA complex formation on DNA replication.
Main Methods:
- Enzymatic assays to measure Topo IV activity in the presence of AP sites.
- Analysis of covalent Topo IV-DNA complex formation and reversal.
- Assessment of helicase activities (DnaB and T7 Gene 4) with AP site-induced complexes.
Main Results:
- AP sites stimulate covalent Topo IV-DNA complex formation at specific positions (4 base overhang and -1 position).
- AP sites function as position-specific endogenous Topo IV poisons.
- Reversal of covalent complexes by EDTA/salt is affected by AP site location; -1 position inhibits reversal.
- AP site-induced complexes do not inhibit DnaB and T7 Gene 4 helicase activities.
Conclusions:
- Abasic sites can act as endogenous poisons for Topoisomerase IV.
- The position of the AP site influences the stability and reversal of Topo IV-DNA complexes.
- AP site-induced Topo IV poisoning does not appear to arrest replication fork progression, unlike drug-induced inhibition.