The Euryarchaeota, Nature's Medium for Engineering of Single-stranded DNA-binding Proteins

Abstract

The architecture of single-stranded DNA-binding proteins, which play key roles in DNA metabolism, is based on different combinations of the oligonucleotide/oligosaccharide binding (OB) fold. Whereas the polypeptide serving this function in bacteria contains one OB fold, the eukaryotic functional homolog comprises a complex of three proteins, each harboring at least one OB fold. Here we show that unlike these groups of organisms, the Euryarchaeota has exploited the potential in the OB fold to re-invent single-stranded DNA-binding proteins many times. However, the most common form is a protein with two OB folds and one zinc finger domain. We created several deletion mutants of this protein based on its conserved motifs, and from these structures functional chimeras were synthesized, supporting the hypothesis that gene duplication and recombination could lead to novel functional forms of single-stranded DNAbinding proteins. Biophysical studies showed that the orthologs of the two OB fold/one zinc finger replication protein A in Methanosarcina acetivorans and Methanopyrus kandleri exhibit two binding modes, wrapping and stretching of DNA. However, the ortholog in Ferroplasma acidarmanus possessed only the stretching mode. Most interestingly, a second single-stranded DNAbinding protein, FacRPA2, in this archaeon exhibited the wrapping mode. Domain analysis of this protein, which contains a single OB fold, showed that its architecture is similar to the functional homologs thought to be unique to the Crenarchaeotes. Most unexpectedly, genes coding for similar proteins were found in the genomes of eukaryotes, including humans. Although the diversity shown by archaeal single-stranded DNA-binding proteins is unparalleled, the presence of their simplest form in many organisms across all domains of life is of greater evolutionary consequence.

Biochemical and Mutational Analyses of a Unique Clamp Loader Complex in the Archaeon Methanosarcina acetivorans

“…However, evidence for how this might have occurred has been elusive. Our effort to systematically assemble the proteins required for replicating the genome of Methanosarcina acetivorans, a mesophilic species of Archaea, has led to several findings of interest to evolution of DNA replication proteins in the archaeal/eukaryotic sister lineages (13)(14)(15). Here, we present the results from biochemical and mutational analyses of an unusual clamp loader found in Methanosarcinales.…”

Role of RPA Proteins in Radiation Repair and Recovery

“…Given the recent identification of a novel SSB in D. radiodurans, and the fundamental role that ssDNA binding proteins play in replication and repair, it is likely that a protein carrying out these essential functions will be found in all organisms. RPAs in euryarchaea present a multitude of OB-fold conformations that presumably provide unique functions (Lin, et al, 2005;Robbins, et al, 2005). Genomes of several euryarchaea, including Thermoplasma acidophilum, Archaeoglobus fulgidus, Ferroplasma acidarmanus, and Halobacterium salinarum, contain genes with similar organization to the crenarchaeal SSB/RPA gene, with one OB-fold; however, in each case, there is also at least one additional homolog that shows different domain structure, as seen in Figure 1 (Komori & Ishino, 2001;Pugh, et al, 2008;Robbins, et al, 2005).…”

“…In H. salinarum, Rfa3, Rfa8, and Ral, and Rfa2 and Rfa7 resemble the P. furiosis RPA with respect to operon structure and sequence homology to eukaryotic RPA (Figure 1). Rfa6 resembles the crenarchaeal SSB/RPA in gene and OB-fold structure, while Rfa1 appears more uniquely archaeal, with three OB-folds (DeVeaux, et al, 2007;Robbins, et al, 2005). The roles of the RPA-like homologs in H. salinarum will be discussed further below.…”

“…RPAs in euryarchaea present a multitude of OB-fold conformations that presumably provide unique functions (Lin, et al, 2005;Robbins, et al, 2005). Genomes of several euryarchaea, including Thermoplasma acidophilum, Archaeoglobus fulgidus, Ferroplasma acidarmanus, and Halobacterium salinarum, contain genes with similar organization to the crenarchaeal SSB/RPA gene, with one OB-fold; however, in each case, there is also at least one additional homolog that shows different domain structure, as seen in Figure 1 (Komori & Ishino, 2001;Pugh, et al, 2008;Robbins, et al, 2005). Other euryarchaea, such as Methanococcus jannaschii, Methanobacter thermoautotrophicus, and the Methanosarcinae, have proteins with four OB-folds, which may act as monomers, or in a complex with an adjacently encoded RPA2 homolog (Chedin, et al, 1998;Kelly, et al, 1998;Komori & Ishino, 2001;Lin, et al, 2008;Robbins, et al, 2004).…”

Correlation Force Spectroscopy for Single Molecule Measurements

“…ssDNA is an essential intermediate in many DNA metabolic processes such as replication, recombination, repair, and transcription, and it is specifically recognized by many DNA-binding proteins, and thus its behavior is of growing interest to the scientific community [124][125][126][127][128][129][130][131][132][133].…”