Super-resolution microscopy can potentially allow imaging of single protein molecules. A new paper now tracks single Pol and Lig proteins in E. coli, as they repair DNA damage.
The researchers replaced the endogenous proteins with proteins tagged with a photoactivatable mCherry (PAmCherry). PAmCherry is non-fluorescent, unless activated by UV light (in this case, a 405 nm laser). By using very short pulses, they activate on average less than one molecule per cell at a time. This method is called photoactivated localization microscopy (PALM).
Using this method, they were able to follow single molecules (see this movie), count the number of Pol & Lig proteins per cell, to measure their binding rates to DNA (at optimal and DNA damage states) and importantly, to measure the timing and rates of the different steps of the base excision repair process (BER).
Their findings show that Pol & Lig diffuse by Brownian motion near undamaged DNA, but immobilize next to damaged DNA. Only a small fraction of the proteins (<5%) are involved in repair under normal conditions. But upon severe damage, the excess proteins come into play, to process >1000 DNA damage sites per minute!
The single proteins fix the damage at about 2 seconds per site. That’s pretty fast!
I think that as PALM and other super-resolution methods become more accessible and prevalent, we will be able to collect amazing data on the function of single proteins and other macro-molecules in vivo.
Uphoff S, Reyes-Lamothe R, Garza de Leon F, Sherratt DJ, & Kapanidis AN (2013). Single-molecule DNA repair in live bacteria. Proceedings of the National Academy of Sciences of the United States of America, 110 (20), 8063-8068 PMID: 23630273