DNA Strand Separation Requires More Force in Crowded Cellular Environments

A new study from Northwestern University suggests that the process of separating DNA strands—a critical step before replication or repair—may require more mechanical force than previously believed when considering the crowded conditions inside living cells.

Traditionally, most biochemistry laboratories study DNA in isolation, using water-based solutions that lack the complexity of a cell’s interior. These labs typically separate DNA strands by heating them to over 150°F, a temperature much higher than what occurs naturally in cells. However, within a living cell, DNA exists in a densely packed environment, surrounded by various molecules and proteins that mechanically unwind and separate the double helix.

John Marko, senior author of the paper published in Biophysical Journal, explained, “The interior of the cell is super crowded with molecules, and most biochemistry experiments are super uncrowded. You can think of extra molecules as billiard balls. They’re pounding against the DNA double helix and keeping it from opening.”

Marko and first author Parth Desai conducted experiments using microscopic magnetic tweezers to separate DNA strands in environments mimicking cellular crowding. By introducing molecules such as glycerol, ethylene glycol, and polyethylene glycol—each similar in size to a DNA double helix—they measured how these agents influenced DNA strand separation. Desai noted, “We wanted to have a wide variety of molecules where some cause dehydration, destabilizing DNA mechanically, and then others that stabilize DNA. It’s not exactly analogous to things found in cells, but you could imagine that other competing proteins in cells will have a similar effect. If they’re competing for water, for instance, they would dehydrate DNA, and if they’re not competing for water, they would crowd the DNA and have this entropic effect.” 

The findings indicate that molecular crowding imposes significant stress on DNA, affecting not only strand separation but potentially all protein interactions with DNA. Marko stated, “If this affects DNA strand separation, all protein interactions with DNA are also going to be affected.”

The team plans further experiments to better replicate the true cellular environment and explore how enzyme-DNA interactions are influenced by crowding.