Molecular biology and biochemistry of archaeal DNA replication
Archaea, together with Bacteria and Eukarya, constitute the three domains of life. In each domain, DNA replication is the process by which members transfer genetic information to offspring to ensure continuity of their lineage. Replication requires a large number of proteins working in concert to faithfully copy the genome, although a level of mutation that leads to either a selective advantage or disadvantage is tolerated. DNA replication is quite well understood in bacteria, but less understood in eukaryotes. Archaea were only discovered in the 1980’s and therefore we know very little of how they replicate their genomes. My lab is interested in assembling the DNA replication apparatus of archaea in vitro. This research theme was motivated by the fascinating discovery that archaeal organisms contain genes coding for proteins with striking similarities to eukaryotic replication proteins. However, the architecture of the archaeal proteins is simpler than the eukaryotic ones. Our ability to assemble an archaeal DNA replication apparatus will therefore not only provide insight into how the genomes in the most recently discovered domain of life are replicated, but will also serve to enhance our understanding of eukaryotic DNA replication including that of humans.
Pyrococcus furiosus is one of the most fascinating archaea, and as its genus name denotes “fireball” this organism grows at temperatures above 100°C. Using this archaeon as a model, we showed that the genes encoding eukaryotic-like DNA replication proteins in archaea indeed code for products that exhibit their predicted biochemical properties. We have further shown that one group of archaea known as the euryarchaeotes, which includes the methanogens, and hyperthermophilic archaea such as Archaeoglobus fulgidus, Thermococcus litoralis, and Pyrococcus furiosus have a unique heterodimeric DNA polymerase currently classified as DNA polymerase family D. From Pyrococcus furiosus, we characterized a clamp (proliferating cell nuclear antigen:PCNA) and a clamp loader complex. These two proteins confer to replicative DNA polymerases, the capacity to rapidly synthesize DNA with infrequent dissociation (processivity), a property required to replicate genomes. The Pyrococcus furiosus clamp loader is the simplest form of the archaeal/eukaryotic clamp loaders, and it has served as a good model for structure/function analysis of clamp loaders. Also, our observation that the P. furiosusclamp is self-loading has very important biotechnological implications, since one can add this stimulatory protein to DNA synthesis reactions containing the P. furiosusDNA polymerase to achieve very rapid synthesis of long products. Furthermore, our work on archaeal DNA polymerases, clamp, and clamp loaders has shown that the mechanism of DNA synthesis at the replication fork is conserved across the three domains of life.
Due to lack of a genetic system for the study of P. furiosus, my lab now usesMethanosarcina acetivorans C2A as our model archaeon. Unlike P. furiosus, M. acetivorans is a mesophilic archaeon that grows optimally at temperatures between 35-40°C. There is a robust genetic system, developed by William Metcalf in our department, for M. acetivorans. This very important tool allows us to study the biochemistry and also the genetics of archaeal DNA replication proteins. We also anticipated that DNA replication in mesophilic archaea may exhibit critical differences from that of hyperthermophilic archaea, and some of our results have already confirmed our hypothesis. For comparative purposes, we also study replication proteins from the hyperthermophilic methanogen (growth temperature 110°C)Methanopyrus kandleri. Our most recent work has concentrated on the biochemistry, biophysical, evolutionary, and structural biology of single-stranded DNA-binding proteins.
We have demonstrated that unlike most organisms, Methanosarcina acetivorans(Mac) and its relatives harbor three different single-stranded DNA-binding proteins. Since these proteins are more similar to their eukaryotic counterparts, we refer to them as replication protein A (RPA) homologs, hence we have characterized MacRPA1, MacRPA2, and MacRPA3. In collaboration with Taekjip Ha’s laboratory in Physics, we have used biophysical methods, mainly Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Polarization Anisotropy (FPA), to study their ssDNA-binding properties, including binding conformation, i.e., whether the RPA proteins bend or stretch DNA upon binding. Based on our studies on RPA proteins, we have come up with two hypotheses in the evolution of RPA proteins, and we have engineered several functional artificial RPA proteins and tested their characteristics through biophysical and biochemical measurements. We also collaborate on single molecule analysis to study the dynamics of clamp loading by the M. acetivoransclamp loader. The M. acetivorans clamp loader is unique and appears to be a “missing link” in the evolution of very complex clamp loaders, as found in humans, from the very simple forms found in P. furiosus. Finally, we have an extensive collaboration with Satish Nair’s lab on structural studies of DNA replication proteins and also on enzymes of plant cell wall hydrolysis. One of the rate-limiting steps in industrial production of cellulosic ethanol is the capacity to hydrolyze cellulosic/hemicellulosic materials to their monomeric forms, such as glucose and xylose, which can then be fermented by yeast to ethanol. My lab is working with Satish Nair’s and Rod Mackie’s labs by using biochemical and structural analyses to engineer cellulases, xylanases, arabinofuranosidases and other polysaccharidases to improve their utility in biofuel production.
A model of an archael clamp loader
An archael replication protein A homolog