Modes of action of the archaeal Mre11/Rad50 DNA-repair complex revealed by fast-scan atomic force microscopy.

Mre11 and Rad50 (M/R) proteins are part of an evolutionarily conserved macromolecular apparatus that maintains genomic integrity through repair pathways. Prior structural studies have revealed that this apparatus is extremely dynamic, displaying flexibility in the long coiled-coil regions of Rad50, a member of the structural maintenance of chromosome (SMC) superfamily of ATPases. However, many details of the mechanics of M/R chromosomal manipulation during DNA-repair events remain unclear. Here, we investigate the properties of the thermostable M/R complex from the archaeon Sulfolobus acidocaldarius using atomic force microscopy (AFM) to understand how this macromolecular machinery orchestrates DNA repair. While previous studies have observed canonical interactions between the globular domains of M/R and DNA, we observe transient interactions between DNA substrates and the Rad50 coiled coils. Fast-scan AFM videos (at 1-2 frames per second) of M/R complexes reveal that these interactions result in manipulation and translocation of the DNA substrates. Our study also shows dramatic and unprecedented ATP-dependent DNA unwinding events by the M/R complex, which extend hundreds of base pairs in length. Supported by molecular dynamic simulations, we propose a model for M/R recognition at DNA breaks in which the Rad50 coiled coils aid movement along DNA substrates until a DNA end is encountered, after which the DNA unwinding activity potentiates the downstream homologous recombination (HR)-mediated DNA repair.

Analysis of phenotype-genotype associations using genomic informational field theory (GIFT).

We show how field- and information theory can be used to quantify the relationship between genotype and phenotype in cases where phenotype is a continuous variable. Given a sample population of phenotype measurements, from various known genotypes, we show how the ordering of phenotype data can lead to quantification of the effect of genotype. This method does not assume that the data has a Gaussian distribution, it is particularly effective at extracting weak and unusual dependencies of genotype on phenotype. However, in cases where data has a special form, (eg Gaussian), we observe that the effective phenotype field has a special form. We use asymptotic analysis to solve both the forward and reverse formulations of the problem. We show how p-values can be calculated so that the significance of correlation between phenotype and genotype can be quantified. This provides a significant generalisation of the traditional methods used in genome-wide association studies GWAS. We derive a field-strength which can be used to deduce how the correlations between genotype and phenotype, and their impact on the distribution of phenotypes.

End-resection at DNA double-strand breaks in the three domains of life.

During DNA repair by HR (homologous recombination), the ends of a DNA DSB (double-strand break) must be resected to generate single-stranded tails, which are required for strand invasion and exchange with homologous chromosomes. This 5'-3' end-resection of the DNA duplex is an essential process, conserved across all three domains of life: the bacteria, eukaryota and archaea. In the present review, we examine the numerous and redundant helicase and nuclease systems that function as the enzymatic analogues for this crucial process in the three major phylogenetic divisions.

Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius.

In eukaryotes, the covalent attachment of ubiquitin chains directs substrates to the proteasome for degradation. Recently, ubiquitin-like modifications have also been described in the archaeal domain of life. It has subsequently been hypothesized that ubiquitin-like proteasomal degradation might also operate in these microbes, since all archaeal species utilize homologues of the eukaryotic proteasome. Here we perform a structural and biochemical analysis of a ubiquitin-like modification pathway in the archaeon Sulfolobus acidocaldarius. We reveal that this modifier is homologous to the eukaryotic ubiquitin-related modifier Urm1, considered to be a close evolutionary relative of the progenitor of all ubiquitin-like proteins. Furthermore we demonstrate that urmylated substrates are recognized and processed by the archaeal proteasome, by virtue of a direct interaction with the modifier. Thus, the regulation of protein stability by Urm1 and the proteasome in archaea is likely representative of an ancient pathway from which eukaryotic ubiquitin-mediated proteolysis has evolved.

Structure of the hexameric HerA ATPase reveals a mechanism of translocation-coupled DNA-end processing in archaea.

The HerA ATPase cooperates with the NurA nuclease and the Mre11-Rad50 complex for the repair of double-strand DNA breaks in thermophilic archaea. Here we extend our structural knowledge of this minimal end-resection apparatus by presenting the first crystal structure of hexameric HerA. The full-length structure visualizes at atomic resolution the N-terminal HerA-ATP synthase domain and a conserved C-terminal extension, which acts as a physical brace between adjacent protomers. The brace also interacts in trans with nucleotide-binding residues of the neighbouring subunit. Our observations support a model in which the coaxial interaction of the HerA ring with the toroidal NurA dimer generates a continuous channel traversing the complex. HerA-driven translocation would propel the DNA towards the narrow annulus of NurA, leading to duplex melting and nucleolytic digestion. This system differs substantially from the bacterial end-resection paradigms. Our findings suggest a novel mode of DNA-end processing by this integrated archaeal helicase-nuclease machine.