Real-time Investigation of SV40 Large T-antigen Helicase Activity Using Surface Plasmon Resonance

Abstract

The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.

G4 Resolvase 1 tightly binds and unwinds unimolecular G4-DNA

“…First, G4R1/RHAU shows significantly more specificity in binding G4-DNA than unstructured single-stranded DNA (Figure 3). In contrast, RPA (52) and T-Ag (54) do not preferentially bind G4 versus unstructured single-stranded DNA. With regard to BLM (52), there is current debate whether this enzyme has a greater specificity for G4-DNA structures versus Watson–Crick duplex DNA.…”

“…G4R1/RHAU also displays a much tighter binding affinity for unimolecular G4-DNA ( K d in the low pM range) than does RPA ( K d in the low µM range) (52), or BLM ( K d of ∼4 nM for binding of tetramolecular G4 DNA (55). The SPR technique could not accurately report a K d for G4-DNA binding by T antigen; however, the working concentration range for T antigen interaction was in the low nanomolar range (54). It is also of interest that G4R1/RHAU tightly binds unimolecular G4-DNA structures without a requirement for divalent cations or ATP.…”

“…It is also of interest that G4R1/RHAU tightly binds unimolecular G4-DNA structures without a requirement for divalent cations or ATP. In contrast, RPA, BLM and T-Ag require divalent cations for G4-DNA binding (52–54); T-Ag requires both Mg 2+ and ATP (54). Although ATP is not required for G4 DNA binding by G4R1/RHAU, the PNA trap demonstrates that ATP is required for G4R1/RHAU-catalyzed G4-DNA resolution.…”

G-quadruplexes in viruses: function and potential therapeutic applications

“…On the other hand, SV40 genome replication requires the large T-antigen (TAg), which is a multifunctional protein that binds the ORI, has adenosine triphosphatedependent helicase activity and interacts with cellular proteins such as p53 and Rb (121). Interestingly, TAg can unwind G4 DNA structures (122,123); thus, it might play a crucial role in regulating replication as well as early and late transcription. Perylene di-imide derivatives (PDI) stabilize G4 structures and inhibit both the G4 and TAg duplex DNA helicase activities.…”

“…Perylene di-imide derivatives (PDI) stabilize G4 structures and inhibit both the G4 and TAg duplex DNA helicase activities. Hence, PDI provide tools for probing the role of the G4 helicase activity in SV40 replication (123,124) and introduces new insights into the link between helicases and tumorigenesis (125) or other human genetic diseases (126).…”