Book of Abstracts: Albany 2009

category image Albany 2009
Conversation 16
June 16-20 2009
© Adenine Press (2008)

Quantifying Multiple DNA Binding Modes of Actinomycin D Using Optical Tweezers

Actinomycin D (Act D) is an antibiotic and antineoplastic compound that has been shown to have significant biological activity, including the ability to inhibit HIV-1 reverse transcription. It is therefore essential to understand the mechanism by which it interacts with nucleic acids. Act D exhibits strong binding to specific sequences of double stranded DNA (dsDNA) and single stranded DNA (ssDNA). However, even after 50 years of research it is not clear which binding mode is strongest. ssDNA binding can be extremely important in inhibiting replication of viruses that replicate through ssDNA templates such as HIV and intercalation can be important in therapeutic application for cancer. DNA stretching studies using optical tweezers can precisely quantify these binding modes. Because both intercalation and ssDNA binding can cause an increase in DNA length observed in these experiments, we have developed a method that combines the measured increase in DNA length with the overall DNA melting free energy change, allowing us to distinguish these binding modes. We determined that the ssDNA binding of ActD (Kss ~ 108 M-1) is 100 fold stronger than its binding to dsDNA (Kds ~ 106 M-1) for long polymeric DNA. The stretching relaxation curve and its hysteresis behavior suggest three different ssDNA binding modes for ActD. In addition, these results suggest a model in which ActD binds to premelted dsDNA and cross stacks with the opposite strand bases. Thus, at saturated binding dsDNA intercalation and ssDNA binding occur simultaneously.

Thayaparan Paramanathan1
Ioana D Vladescu2
Micah J. McCauley1
Ioulia Rouzina3
Mark C.Williams1

1Department of Physics
Northeastern University
Boston, MA, USA
2Harvard FAS Center
for Systems Biology
Harvard University
Cambridge, MA, USA
3Dept. of Biochemistry
Molecular Biology and Biophysics
University of Minnesota
Minneapolis, MN USA