DNA repair protein is a three-way zipper

New work from UC Davis explains how a protein motor named Rad54 helps both to form and to break down the “displacement loop,” or D-loop, a key intermediate in the important DNA repair pathway, homologous recombination.

Formation of the D-loop allows a cell to use a broken or damaged DNA molecule to locate and synthesize lost sequence from an intact “donor” DNA molecule containing the same sequence. The cell needs to do this in order to maintain its genome, and it is therefore of crucial importance in understanding the origins of cancer and birth defects.

“It’s a big step for the field because it’s been unclear what Rad54 does,” said Wolf-Dietrich Heyer, professor in the Departments of Microbiology and Molecular Genetics and of Molecular and Cellular Biology in the College of Biological Sciences and at the UC Davis Comprehensive Cancer Center. Heyer is coauthor of the paper published Jan. 30 in the journal Molecular Cell, along with graduate student William Douglass Wright.

Before DNA can be copied from the donor molecule, the damaged and donor DNA molecules have to find each other. A protein called Rad51 is responsible for lining a DNA strand and stretching it like a spring, so that the bases that make up the genetic code (A, C, G and T) can flip outwards.

During the search for the template from which to copy the missing information, Rad51 also stretches the donor DNA to match it to the broken DNA forming a unique three-stranded structure, the D-loop.

It’s been known that Rad54 both assists Rad51 in forming the D-loop and also disrupts D-loops, but until now it has not been clear how that happens.

Wright used purified DNA molecules with filaments of Rad51 to show that Rad54 acts as a pump that pushes a strand of the damaged DNA and a strand of the donor molecule through itself to create double-stranded DNA, knocking off Rad51 as it does so. Rad54 moves along DNA in the direction in which it detects Rad51.

In other words, presented with three-stranded DNA stretched open by Rad51, Rad54 moves along it, knocking out the Rad51 so that the structure can snap back into its normal form. Two strands are pushed together, but at the same time, another part of the Rad54 protein is in contact with the third strand of DNA, which is displaced.

graph abstract1So Rad54 is acting as a three-way zipper: it brings together two strands of DNA, while at the same time it separates a third strand of DNA that would otherwise interfere.

“This model consolidates both Rad51 removal and DNA zippering functions into one step,” Heyer said. It also helps place Rad54 in a class of other proteins that act as DNA pumps, for example during bacterial cell division.

The work was supported by grants from the National Institutes of Health, and a fellowship to Wright from the Tobacco-Related Disease Research Program.

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