Kathleen Sandman

Diversity of prokaryotic chromosomal proteins and the origin of the nucleosome.

Abstract. All cells employ architectural proteins to confine and organize their chromosomes, and to prevent the otherwise thermodynamically favored collapse of concentrated DNA into compact structures. To accomplish this, prokaryotes have evolved a variety of phylogenetically unrelated, small, basic, sequence-independent DNA-binding proteins that include histones in Euryarchaeota, and members of the HU family in many Bacteria. In contrast, virtually all Eukarya employ histones, and recently a metabolism-based hypothesis proposed that the eukaryal nucleus originated from a hydrogen-consuming, histone-containing Archaeon. Histones may have prevailed during the evolution of the Eukarya because of their extended interactions with DNA and, as noted, the histone fold now exists not only in histones but also as a structural motif in eukaryal transcription factors.

Histones in Crenarchaea

Archaeal histone-encoding genes have been identified in marine Crenarchaea. The protein encoded by a representative of these genes, synthesized in vitro and expressed in Escherichia coli, binds DNA and forms complexes with properties typical of an archaeal histone. The discovery of histones in Crenarchaea supports the argument that histones evolved before the divergence of Archaea and Eukarya.

DNA stability and DNA binding proteins.

Publisher Summary This chapter reviews the initial studies that focus on characterizing the factors that contribute to the stability of the genomes of hyperthermophiles, both extrinsic factors, such as the intracellular ionic environment and so-called histone like DNA binding proteins, and intrinsic factors, such as DNA base composition and topology. Hyperthermophiles probably also have very efficient DNA repair mechanisms, but research in this area has been limited, although initial characterizations of red homologs, a photolyase, and superoxide dismutases have been reported. The chapter discusses thermal degradation and chemical degradation at high temperature of DNA. Both eukaryotes and prokaryotes also contain DNA binding proteins that are clearly involved in genome compaction. These abundant structural proteins contribute to both the local and global architecture of genomes, but they also participate directly in regulating gene expression, and most probably in thermophiles, also in genome thermostabilization. During DNA replication, recombination, and transcription, the topology of a circular DNA molecule change, and topoisomerases are employed to adjust and stabilize the topology of the circular genomes of prokaryotes.

Histone-encoding genes from Pyrococcus: evidence for members of the HMf family of archaeal histones in a non-methanogenic Archaeon

Two genes, designated hpyA1 and hpyA2, have been cloned and sequenced from Pyrococcus strain GB-3a. They are predicted to encode proteins (HPyA1 and HPyA2, respectively) that are approx. 60% identical to the histones HMf and HMt, characterized from methanogenic Archaea. These archaeal histones also contain the amino-acid sequences, conserved in eukaryotic H4 histones, that are thought to interact directly with DNA.

Archaeal histone stability, DNA binding, and transcription inhibition above 90°C

Abstract The DNA binding and compacting activities of the recombinant (r) archaeal histones rHMfA and rHMfB from Methanothermus fervidus, and rHPyA1 from Pyrococcus species GB-3a, synthesized in Escherichia coli, have been shown to be completely resistant to incubation for 4 h at 95°C in the presence of 1 M KCl. Continued incubation of rHMfA and rHMfB at 95°C resulted in a gradual loss of these activities, and rHMfA and rHMfB lost activity more rapidly at 95°C when the salt environment was reduced to 200 mM KCl. rHPyA1, in contrast, retained full activity even after a 60-h incubation at 95°C in 1 M KCl, and reducing the salt concentration did not affect the heat resistance of rHPyA1. rHPyA1–DNA complexes remained intact at 100°C, and rHPyA1 bound to the template DNA in in vitro transcription reaction mixtures assembled using Pyrococcus furiosus components at 90°C. Transcription in vitro from the P. furiosus gdh promoter was reduced by rHPyA1 binding, in a manner that was dependent on the histone-to-DNA ratio and on the topology of the DNA template. Transcription from circular templates was more sensitive to rHPyA1 binding than transcription from a linear template, consistent with rHPyA1 binding introducing physical barriers to transcription and causing changes in the topology of circular templates that also reduced transcription.

Deletion of the archaeal histone in Methanosarcina mazei Gö1 results in reduced growth and genomic transcription

HMm is the only archaeal histone in Methanosarcina mazei Göl and recombinant HMm, synthesized by expression of MM1825 in Escherichia coli, has been purified and confirmed to have the DNA binding and compaction properties characteristic of an archaeal histone. Insertion of a puromycin resistance conferring cassette (pac) into MM1825 was not lethal but resulted in mutants (M. mazei MM1825::pac) that have impaired ability to grow on methanol and trimethylamine. Loss of HMm also resulted in increased sensitivity to UV light and decreased transcript levels for ∼25% of all M. mazei genes. For most genes, the transcript decrease was 3‐ to 10‐fold, but transcripts of MM483 (small heat‐shock protein), MM1688 (trimethylamine:corrinoid methyl transferase) and MM3195 (transcription regulator), were reduced 100‐, 100‐ and 25‐fold, respectively, in M. mazei MM1825::pac cells. Transcripts of only five adjacent genes that appear to constitute an aromatic amino acid biosynthetic operon were elevated in M. mazei MM1825::pac cells. Complementary synthesis of HMm from a plasmid transformed into M. mazei MM1825::pac restored wild‐type growth and transcript levels.

Archaeal DNA Binding Proteins and Chromosome Structure

Summary Small, basic, abundant DNA-binding proteins have been isolated from many different prokaryotes. Proteins of this type that have been characterized from Archaea are reviewed here, and their structural and functional relationships to bacterial histone-like proteins and eukaryal histones are discussed. Members of the HMf family of archaeal DNA-binding proteins are considered most similar to eukaryal histones and it is proposed that in Methanothermus fervidus cells, which normally contain a high internal salt concentration and grow at temperatures above 80 °C, that HMf both compacts the genomic DNA and facilitates its replication and expression.

[10] Archaeal histones and nucleosomes

Publisher Summary Almost all eukarya employ histones, small basic proteins that organize DNA into nucleosomes, which are further assembled into chromatin and chromosomes. Prokaryotic homologs of the nucleosome have been documented in the Euryarchaeota, one branch of the Domain Archaea. Archaeal and eukaryal histones share a common evolutionary ancestry: they exhibit both amino acid sequence similarity and a conserved three-dimensional histone-fold structure. Numerous techniques have been developed for the experimental manipulation of eukaryal histones and nucleosomes, but most are not applicable to their prokaryotic counterparts. This chapter provides protocols for the purification of archaeal histones and nucleosomes, and procedures for assays of DNA binding and archaeal nucleosome positioning.

Structure of histone-based chromatin in Archaea

Origin of DNA compaction As a repeating unit in eukaryotic chromatin, a nucleosome wraps DNA in superhelical turns around a histone octamer. Mattiroli et al. present the crystal structure of an archaeal histone-DNA complex in which the histone-mediated DNA geometry is exactly the same as that in the nucleosome. Comparing features of archaeal and eukaryotic chromatin structures offers important insights into the evolution of eukaryotic nucleosomes. Science, this issue p. 609 Archaeal histone homodimers form a complex with DNA that is similar to the eukaryotic nucleosome. Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.

Molecular components of the archaeal nucleosome.

Here we describe the organization of the archaeal nucleosome, in which four archaeal histones are circumscribed by approximately 80 bp of DNA. Through a combination of sequence comparisons, 3D structural studies, site-directed mutagenesis and assays for DNA binding, we have assigned functions to most of the individual residues in the histone fold of the representative archaeal histone rHMfB. By SELEX selection, the sequences of DNA molecules that are most readily bound and wrapped by rHMfB into archaeal nucleosomes in vitro have been identified, and these define DNA structures that position archaeal nucleosome assembly.