For many years, my lab has been interested in understanding the molecular basis of DNA replication in archaea. Part of our motivation to do so lies in the fact that archaea are fascinating and under-studied organisms. But we’re also inspired by the close evolutionary relationship between archaea and eukaryotes. In particular, the information processing machineries of archaea are in essence a simplified form of those in eukaryotes.

A lot of our early work was focused on investigating the roles of homologs of known eukaryotic replication proteins. However, we have, of course, been aware that, with a 2 billion year separation between archaea and eukaryotes, there must have been innovations in the archaeal lineages and embellishment of the shared core machinery with archaeal-specific “dark matter” proteins – proteins required for replication that lack clear homologs in eukaryotes (or even in bacteria). How then do we identify that dark matter?

In a new paper in Nature Communications we describe the identification and characterization of one such protein, UBP. The story began back in the early 2000s when Nick Robinson, then a post-doc in my lab,  spotted a conserved sequence motif in origins of DNA replication in several members of the crenarchaea. Nick’s work, in parallel with work from the late Rolf Bernander’s lab, had revealed that archaea can possess multiple origins of DNA replication per chromosome. Nick, through sequence gazing, had spotted a short motif shared between the origins and we termed it the uncharacterized motif (ucm) because that’s what it was!

Shortly after this, my lab relocated to Oxford and a new post-doc, Alessandro Costa, was interested in working out what the ucm was doing. We reasoned it was likely to be a binding site for an unknown factor and through a combination of conventional and DNA-affinity chromatography, Alessandro succeeded in purifying the candidate protein, which we termed UBP for ucm-binding protein. UBP was novel protein, with no homologs in either eukaryotes or bacteria. He did some nice biochemistry investigating the interaction of UBP with DNA but what role, if any, it played in replication remained unknown.

The lab moved again, this time to Indiana in the US and another post-doc Xu Feng joined the lab, bringing skills in the genetic manipulation of archaea with him. Harnessing the endogenous Sulfolobus CRISPR system, Xu performed scanning mutagenesis of one of the origins of replication and using these mutant lines, Xu and another post-doc in the lab, Rachel Samson, revealed that the integrity of the ucm was absolutely essential for origin function.

Xu found that UBP interacted with the replicative MCM helicase but also made use of an in vitro helicase loading assay developed in the lab to show that UBP played a very minor role in recruiting MCM. About this time, Rachel, using chromatin immunoprecipitation approaches, made the striking discovery that in the absence of the ucm, and in absence of UBP binding to the origin, MCM was still recruited to the origin but was present at significantly elevated levels compared to the wild-type origin.

Thus, it appeared that UBP was not a recruitment factor, but rather played a key role in facilitating departure of MCM from the replication origin. So, we now had many pieces of the puzzle and at this time Raj Dhanaraju joined the lab as a grad student. Working with Giovanni Gonzalez-Gutierrez in the outstanding macromolecular crystallography facility at IU Bloomington, which was down the corridor from our lab, they solved the crystal structures of UBP in the presence and absence of DNA. This revealed a rather cute strand-swap dimerization mode of the unbound UBP and a transition to monomer upon interaction with the ucm.

Another level to the significance of UBP emerged about this time – Raj had quantified the protein in cells and found it to be rather abundant and Rachel Samson performed ChIP-Seq studies and revealed that not only did UBP bind specifically to the replication origins but it also bound to hundreds of other loci around the chromosome. A genome-wide analysis by Rachel Whitaker’s lab had shown the ubp gene to be essential for viability in Sulfolobus so we couldn’t knock it out, but Raj succeeded in over-expressing the protein from a plasmid.

Intriguingly, this led to extended growth of the over-expressing cultures and a delayed entry into stationary phase. Raj performed RNA-Seq and we observed that over-expression of UBP led to an extended execution of the gene expression profile associated with rapidly growing cells.

So, taken together, our data support a model in which UBP is a novel, nucleoid-associated protein in Sulfolobus. It influences expression of a range of genes and may play a role in maintaining a gene expression profile compatible with rapid growth. It also plays a pivotal role at DNA replication origins, where our data support a model of UBP helping the MCM helicase exit from the origin during the replication initiation process.

It’s been a long trip, from the initial purification almost 18 years ago, to gain some molecular insight into UBP’s function. We hope for considerably more rapid progress as we dissect the gene-expression and replication-associated functions of this intriguing piece of archaeal dark matter.