Divalent metal cofactors differentially modulate RadA-mediated strand invasion and exchange in Saccharolobus solfataricus.

Central to the universal process of recombination, RecA family proteins form nucleoprotein filaments to catalyze production of heteroduplex DNA between substrate ssDNAs and template dsDNAs. ATP binding assists the filament in assuming the necessary conformation for forming heteroduplex DNA, but hydrolysis is not required. ATP hydrolysis has two identified roles which are not universally conserved: promotion of filament dissociation and enhancing flexibility of the filament. In this work, we examine ATP utilization of the RecA family recombinase SsoRadA from Saccharolobus solfataricus to determine its function in recombinase-mediated heteroduplex DNA formation. Wild-type SsoRadA protein and two ATPase mutant proteins were evaluated for the effects of three divalent metal cofactors. We found that unlike other archaeal RadA proteins, SsoRadA-mediated strand exchange is not enhanced by Ca2+. Instead, the S. solfataricus recombinase can utilize Mn2+ to stimulate strand invasion and reduce ADP-binding stability. Additionally, reduction of SsoRadA ATPase activity by Walker Box mutation or cofactor alteration resulted in a loss of large, complete strand exchange products. Depletion of ADP was found to improve initial strand invasion but also led to a similar loss of large strand exchange events. Our results indicate that overall, SsoRadA is distinct in its use of divalent cofactors but its activity with Mn2+ shows similarity to human RAD51 protein with Ca2+.

The RadA Recombinase and Paralogs of the Hyperthermophilic Archaeon Sulfolobus solfataricus.

Repair of DNA double-strand breaks is a critical function shared by organisms in all three domains of life. The majority of mechanistic understanding of this process has come from characterization of bacterial and eukaryotic proteins, while significantly less is known about analogous activities in the third, archaeal domain. Despite the physical resemblance of archaea to bacteria, archaeal proteins involved in break repair are remarkably similar to those used by eukaryotes. Investigating the function of the archaeal version of these proteins is, in many cases, simpler than working with eukaryotic homologs owing to their robust nature and ease of purification. In this chapter, we describe methods for purification and activity analysis for the RadA recombinase and its paralogs from the hyperthermophilic acidophilic archaeon Sulfolobus solfataricus.

An archaeal RadA paralog influences presynaptic filament formation.

Recombinases of the RecA family play vital roles in homologous recombination, a high-fidelity mechanism to repair DNA double-stranded breaks. These proteins catalyze strand invasion and exchange after forming dynamic nucleoprotein filaments on ssDNA. Increasing evidence suggests that stabilization of these dynamic filaments is a highly conserved function across diverse species. Here, we analyze the presynaptic filament formation and DNA binding characteristics of the Sulfolobus solfataricus recombinase SsoRadA in conjunction with the SsoRadA paralog SsoRal1. In addition to constraining SsoRadA ssDNA-dependent ATPase activity, the paralog also enhances SsoRadA ssDNA binding, effectively influencing activities necessary for presynaptic filament formation. These activities result in enhanced SsoRadA-mediated strand invasion in the presence of SsoRal1 and suggest a filament stabilization function for the SsoRal1 protein.

Repair of DNA double-strand breaks induced by ionizing radiation damage correlates with upregulation of homologous recombination genes in Sulfolobus solfataricus.

The mechanisms used by members of the archaeal branch of life to repair DNA damage are not well understood. DNA damage responses have been of particular interest in hyperthermophilic archaea, since these microbes live under environmental conditions that constantly elevate the potential for DNA damage. The work described here focuses on the response of four Sulfolobus solfataricus strains to ionizing radiation (IR) damage. Cellular survival of three wild-type strains and a defined deletion mutant strain was examined following exposure to various IR doses. Using pulsed-field gel electrophoresis, we determined chromosomal DNA double-strand break persistence and repair rates. Among the strains, variable responses were observed, the most surprising of which occurred with the defined deletion mutant strain. This strain displayed higher chromosomal repair rates than the parent strain and was also found to have increased resistance to IR. Using quantitative real-time PCR, we found that transcript levels of homologous recombination-related genes were strongly upregulated following damage in all the strains. The mutant strain again had an enhanced response and dramatically upregulated expression of recombination genes above levels observed for the parent strain, suggesting that increased levels of recombinational repair could account for its increased radiation resistance phenotype. Our results demonstrate a transcriptional response to IR in S. solfataricus for the first time and describe a defined deletion mutant strain that may give the first insight into a damage-based archaeal control element.

Repair of DNA double-strand breaks following UV damage in three Sulfolobus solfataricus strains.

DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for double-strand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage.

The single-stranded DNA binding protein of Sulfolobus solfataricus acts in the presynaptic step of homologous recombination.

Homologous recombination is an important pathway in the repair of DNA double-strand breaks in all organisms. In mesophiles, single-stranded DNA binding proteins (SSBs) are believed to be involved in the removal of single-stranded DNA (ssDNA) secondary structure during the presynaptic step of homologous recombination, facilitating the formation of a contiguous Rad51/RecA nucleoprotein filament. Here we report a role for the thermophilic archaeal Sulfolobus solfataricus SSB (SsoSSB) in the presynaptic step of homologous recombination. We have identified multiple quaternary structural forms of this protein in vivo and examined the activity of SsoSSB with the strand-exchange protein S. solfataricus RadA (SsoRadA). Using gel-shift analysis, we found that the two major forms of SsoSSB have different DNA binding affinities and site sizes. Biochemical examination of the monomeric form of SsoSSB suggests that it has a minor role in presynapsis and may slightly inhibit the ssDNA-dependent ATPase activity of SsoRadA. The tetrameric form of SsoSSB, however, significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Order-of-addition experiments indicate that preincubation of tetrameric SsoSSB and SsoRadA prior to reaction initiation with ssDNA relieves the inhibition observed when SsoSSB is added either before or after SsoRadA. In addition, we demonstrate a direct interaction between SsoRadA and SsoSSB using coimmunoprecipitation. Taken together, these results suggest that a direct interaction between SsoSSB and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament.

Identification of RTG2 as a modifier gene for CTG*CAG repeat instability in Saccharomyces cerevisiae.

Trinucleotide repeats (TNRs) undergo frequent mutations in families affected by TNR diseases and in model organisms. Much of the instability is conferred in cis by the sequence and length of the triplet tract. Trans-acting factors also modulate TNR instability risk, on the basis of such evidence as parent-of-origin effects. To help identify trans-acting modifiers, a screen was performed to find yeast mutants with altered CTG.CAG repeat mutation frequencies. The RTG2 gene was identified as one such modifier. In rtg2 mutants, expansions of CTG.CAG repeats show a modest increase in rate, depending on the starting tract length. Surprisingly, contractions were suppressed in an rtg2 background. This creates a situation in a model system where expansions outnumber contractions, as in humans. The rtg2 phenotype was apparently specific for CTG.CAG repeat instability, since no changes in mutation rate were observed for dinucleotide repeats or at the CAN1 reporter gene. This feature sets rtg2 mutants apart from most other mutants that affect genetic stability both for TNRs and at other DNA sequences. It was also found that RTG2 acts independently of its normal partners RTG1 and RTG3, suggesting a novel function of RTG2 that helps modify CTG.CAG repeat mutation risk.