Unraveling Reversible DNA Cross-Links with a Biological Machine

Abstract

The reversible generation and capture of certain electrophilic quinone methide intermediates support dynamic reactions with DNA that allow for migration and transfer of alkylation and cross-linking. This reversibility also expands the possible consequences that can be envisioned when confronted by DNA repair processes and biological machines. To begin testing the response to such an encounter, quinone methide-based modification of DNA has now been challenged with a helicase (T7 bacteriophage gene protein four, T7gp4) that promotes 5' to 3' translocation and unwinding. This model protein was selected based on its widespread application, well characterized mechanism and detailed structural information. Little over one-half of the cross-linking generated by a bisfunctional quinone methide remained stable to T7gp4 and did not suppress its activity. The helicase likely avoids the topological block generated by this fraction of cross-linking by its ability to shift from single- to double-stranded translocation. The remaining fraction of cross-linking was destroyed during T7gp4 catalysis. Thus, this helicase is chemically competent to promote release of the quinone methide from DNA. The ability of T7gp4 to act as a Brownian ratchet for unwinding DNA may block recapture of the QM intermediate by DNA during its transient release from a donor strand. Most surprisingly, T7gp4 releases the quinone methide from both the translocating strand that passes through its central channel and the excluded strand that was typically unaffected by other lesions. The ability of T7gp4 to reverse the cross-link formed by the quinone methide does not extend to that formed irreversibly by the nitrogen mustard mechlorethamine.

Figure 1.

Unwinding DNA and release of a reversible interstrand cross-link. (A) A replication fork model (5′-[³²P]-OD1:OD2, 10 nM) containing an interstrand cross-link formed by bisQMP and a radiolabel on the translocated strand was incubated in the alternative and combined presence of T7gp4 (55 nM monomer) and dTTP (1 mM). (B) After incubation at 37 °C for the indicated time, reaction was quenched by addition of EDTA (40 mM) and analyzed by denaturing polyacrylamide gel electrophoresis (PAGE, 10%). As electrophoretic standards, lane ss contains only 5′-[³²P]-OD1 and lane xl contains 5′-[³²P]-OD1:OD2 cross-linked by bisQMP. (C) The remaining cross-link (% based on total radiolabel) was determined by phosphoimagery in three replicates and their average values are indicated by the cross-bars.

Figure 2.

Supplementing conditions after initial unwinding of DNA containing a reversible interstrand cross-link. (A) A second replication fork model (OD3:5′-[³²P]-OD4, 10 nM) containing a cross-link formed by bisQMP and a radiolabel on the excluded strand was incubated as described in Figure 1 for 30 min before supplementing with another aliquot of either T7gp4 (55 nM monomer) or dTTP (1 mM). Incubations were continued at 37 °C for the indicated times and then quenched with EDTA (40 mM). (B) Reaction products were detected after separation by denaturing PAGE (10%) and quantified relative to total radiolabel (%). Data represent three replicates and average values are indicated by the cross-bars. The control entitled spontaneous hydrolysis contained no T7gp4 or dTTP throughout the incubations and that entitled no supplement contained the standard concentrations of T7gp4 and dTTP in the initial incubation but no additional T7gp4 or dTTP in the subsequent incubation.

Figure 3.

Challenging T7gp4 with an irreversible cross-link. (A) The replication fork model (OD3:5′-[³²P]-OD4) containing a cross-link formed by mechlorethamine and a radiolabel on the excluded strand was treated in the alternative and combined presence of T7gp4 and dTTP as described in Figure 1. (B) Reaction products were detected after separation by denaturing PAGE (10%) and quantified relative to total radiolabel (%). Data represent three replicates and average values are indicated by the cross-bars.

Figure 4.

Unwinding DNA and release of a reversible DNA adduct. (A) A model replication fork (5′-[³²P]-OD3:OD4, 10 nM) containing reversible adducts formed by monoQMP and a radiolabel on the translocated strand was incubated in the alternative and combined presence of T7gp4 (55 nM monomer) and dTTP (1 mM). (B) After incubation at 37 °C for the indicated time, reaction was quenched by addition of EDTA (40 mM) and analyzed by denaturing PAGE (10%). As electrophoretic standards, lane ss contains only 5′-[³²P]-OD3 and lane alk contains OD3 after treatment with monoQMP. (C) The remaining cross-link (% based on total radiolabel) was measured by phosphoimagery in three replicates and their average values are indicated by the cross-bars.

Scheme 1.

(A) Quinone Methide Deprotection, Generation, and Reversible Reaction with DNA and (B) Bifunctional and Monofunctional Quinone Methide Precursors

Scheme 2.

Dynamic Migration of Intra- and Interstrand Cross-Linking by a Bisfunctional and Reversible QM

Scheme 3.

Confrontation between a Helicase and a Reversible Cross-Link May Have Varied Consequences

Scheme 4.

Reversible Alkylation May Persist or Release during DNA Unwinding