Single molecule measurements of mitochondrial protein-DNA interactions

Tuesday, October 09, 2012 — Poster Session I
1:00 p.m. – 3:00 p.m	Natcher Conference Center, Building 45	NHLBI	BIOPHY-5

Authors

• TR Litwin
• IJ Holt
• KC Neuman

Abstract

Mitochondria are essential to the functioning of eukaryotic organisms. They contain a circular 16.6 kb genome (mtDNA) that encodes rRNA, tRNA, and thirteen essential proteins. The mtDNA is associated with a set of nucleoid proteins that package, organize, and protect it. One nucleoid protein is ATAD3, an AAA+ ATPase that supports mitochondrial protein synthesis. siRNA knockdown of ATAD3 decreases the amount of mtDNA by about 15% and induces a large change, presumed to be topological, that prevents visualization of the remaining mtDNA using PicoGreen intercalating dye. To investigate this phenomenon we use magnetic tweezers to probe the effect of PicoGreen and ATAD3 binding on lambda DNA tension length and topology. Magnetic tweezers are a single molecule technique in which mechanical stress on individual DNA molecules, for instance that resulting from PicoGreen or ATAD3 binding, can be measured with high precision. Another core nucleoid protein is Tfam (mitochondrial transcription factor A), a protein which coordinates nucleoid assembly and regulates mtDNA copy number. It also imposes a strong bend on DNA and has been reported to cause negative supercoiling. Magnetic tweezers will be used to investigate binding site size, DNA bend angle, and the topological consequences of Tfam-DNA interactions.

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