Molecular DNA switch found to be the same for all life

The molecular machinery that starts the process by which a biological cell divides into two identical daughter cells apparently worked so well early on that evolution has conserved it across the eons in all forms of life on Earth.

Researchers with the US Department of Energy's Lawrence Berkeley National Laboratory and the University of California at Berkeley have shown that the core machinery for initiating DNA replication is the same for all three domains of life Archaea, Bacteria and Eukarya.

In two papers that will be published in the August edition of the journal Nature Structural and Molecular Biology, the researchers report the identification of a helical substructure within a superfamily of proteins, called AAA+, as the molecular "initiator' of DNA replication in a bacteria, E.coli, and in a eukaryote, Drosophila melanogaster, the fruit fly.

Taken with earlier research that identified AAA+ proteins at the heart of the DNA replication initiator in archaea organisms, these findings indicate that DNA replication is an ancient event that evolved millions of years ago, prior to when archaea, bacteria and eukarya split into separate domains of life.

"The ability of a cell to replicate its DNA in a timely and faithful manner is fundamental for survival, but despite decades of study, the structural and molecular basis for initiating DNA replication, and the degree to which these mechanisms have been conserved by evolution, have been ill defined and hotly debated," said biophysicist Eva Nogales, a collaborator on the Drosophila study.

"Our two papers fuse together a number of biophysical research techniques to take our understanding of the mechanics of DNA opening and replisome construction to a new level," said biochemist Michael Botchan, also a collaborator on the Drosophila study.

"Our findings of evolutionary kinship between the DNA initiators in all three domains make sense because, to paraphrase Francois Jacob, the one thing a cell wants to do is to become two cells. A cell can't do this if it doesn't replicate its DNA in the right place, at the right time, and in the right manner, while simultaneously avoiding over-replication," said biochemist and structural biologist James Berger, a participant in both studies.

The Drosophila results were reported in a paper entitled: "Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex'.

The E.coli results were presented in a paper entitled: "Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling'.

For the E.coli study, Berger and his team used the exceptionally bright and intense x-rays of Beamline 8.3.1 at Berkeley Lab's Advanced Light Source synchrotron. With the data gathered at this protein crystallography facility, Berger and his team assembled a high-resolution model of the molecular structure of a protein known as DnaA, which is a member of the AAA+ family.

While it has long been known that DnaA controls the process of initiating DNA replication in bacteria, the molecular details of its myriad activities have until now been a mystery.

Berger's team found that when the DnaA protein binds with adenosine triphosphate or ATP, the nucleotide molecule that supplies energy to all components of a cell, the ring-shaped AAA+ proteins assemble into a right-handed spiralling superstructure.

This arrangement was unexpected, because in other functional AAA+ complexes, the ring assemblies are closed. In addition, the architecture indicated that the AAA+ super-helix will wrap coils of the DNA double-helix around its exterior, causing the familiar "spiral staircase' of the DNA to deform as a first step in the separation and unwinding of its two gene-carrying strands.

"It is likely that the AAA+ rings of the replication initiators are open to allow other proteins to dock onto the initiator complex," said Berger.

"These other proteins can help add layers of complexity, such as assisting with helicase loading or inactivating the initiator after replication has begun. The open rings also probably allow DNA to interact with the interior of the initiator assembly."

Bacterial cells, like the cells of Achaeans, are prokaryotes, meaning their DNA is not contained within a defined nucleus. Eukaryotes consist of plants and animals and all other organisms whose DNA is contained within a membrane-bound nucleus.

Whereas DNA replication in bacteria is typically initiated at a single site, DNA replication in eukaryotes can be an immensely complicated multi-event affair, involving the coordinated initiation and regulation of hundreds and even thousands of protein machines at different sites throughout the genome.

Furthermore, the highly packaged nature of eukaryotic genomes makes it difficult for these protein machines to access the DNA. Because of this complexity, the mechanism for initiating DNA replication in eukaryotes was presumed to be much different than the prokaryote initiator.

"This work provides the first view of the mechanical transitions in ORC driven by ATP in a higher organism," said Nogales.

"While our studies have not yet shown the initiator wrapped around the DNA, the structural similarity to the DnaA initiator found in the E.coli study suggests that there are likely to be strong mechanistic commonalities in the ways that initiators engage and remodel replication origins, as well as in how they facilitate replisome assembly."

The idea that all three domains of life share the same DNA replication initiator is new and will require some re-thinking on the part of biologists who study eukaryotes. Re-thinking will also be required for models of DNA replication that predicted initiators would have similar structures to the protein "clamps' and "clamp loaders' already identified as key mechanisms in the DNA replication process.

"Our work shows that there are major structural distinctions between assembled initiator and clamp loader complexes. This not only has important implications for the respective functions of these different mechanisms, it also calls into question some cherished models in the field," said Berger.

The two studies by Nogales, Berger, Botchan and their colleagues also show how when nature finds a mechanism that works well, such a mechanism is conserved through evolution.

"The specialisation of DNA replication initiators took place a long time ago, separating them from other members of the AAA+ superfamily of proteins while maintaining an identity among themselves that reflects the importance of the replication process. Through the millions of years, evolution has added bells and whistles around this highly conserved central engine," said Nogales.

For further information, visit Lawrence Berkeley National Laboratory

Copies of the two scientific papers discussed in this press release can be viewed online at Nature structural and molecular biology