Insights into the evolutionary conserved regulation of Rio ATPase activity.

Eukaryotic ribosome biogenesis is a complex dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1, Rio2 and Rio3) belong to an ancient conserved atypical protein kinase/ ATPase family required for the maturation of the small ribosomal subunit (SSU). Recent structure-function analyses suggested an ATPase-dependent role of the Rio proteins to regulate their dynamic association with the nascent pre-SSU. However, the evolutionary origin of this feature and the detailed molecular mechanism that allows controlled activation of the catalytic activity remained to be determined. In this work we provide functional evidence showing a conserved role of the archaeal Rio proteins for the synthesis of the SSU in archaea. Moreover, we unravel a conserved RNA-dependent regulation of the Rio ATPases, which in the case of Rio2 involves, at least, helix 30 of the SSU rRNA and the P-loop lysine within the shared RIO domain. Together, our study suggests a ribosomal RNA-mediated regulatory mechanism enabling the appropriate stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent pre-40S particle. Based on our findings we propose a unified release mechanism for the Rio proteins.

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the ß7-ß8 and ß9-ß10 hairpin loops and conserved zinc binding domain cysteines.

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the ß7-ß8 and ß9-ß10 ß-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the ß7-ß8 ß-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the ß7-ß8 ß-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the ß9-ß10 ß-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the ß9-ß10 ß-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environment.

The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from the halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 A resolution to a final R factor of 23.7% (R(free) = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.

Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP-binding site and arginine finger motif.

Replication factor C (RFC) plays a key role in eukaryotic chromosome replication by acting as a loading factor for the essential sliding clamp and polymerase processivity factor, proliferating cell nuclear antigen (PCNA). RFC is a pentamer comprising a large subunit, Rfc1, and four small subunits, Rfc2-Rfc5. Each RFC subunit is a member of the AAA+ family of ATPase and ATPase-like proteins, and the loading of PCNA onto double-stranded DNA is an ATP-dependent process. Here, we describe the properties of a collection of 38 mutant forms of the Rfc2 protein generated by pentapeptide-scanning mutagenesis of the fission yeast rfc2 gene. Each insertion was tested for its ability to support growth in fission yeast rfc2Delta cells lacking endogenous Rfc2 protein and the location of each insertion was mapped onto the 3D structure of budding yeast Rfc2. This analysis revealed that the majority of the inactivating mutations mapped in or adjacent to ATP sites C and D in Rfc2 (arginine finger and P-loop, respectively) or to the five-stranded beta sheet at the heart of the Rfc2 protein. By contrast, nonlethal mutations map predominantly to loop regions or to the outer surface of the RFC complex, often in highly conserved regions of the protein. Possible explanations for the effects of the various insertions are discussed.

Functional mapping of the fission yeast DNA polymerase delta B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis.

BACKGROUND: DNA polymerase delta plays an essential role in chromosomal DNA replication in eukaryotic cells, being responsible for synthesising the bulk of the lagging strand. In fission yeast, Pol delta is a heterotetrameric enzyme comprising four evolutionarily well-conserved proteins: the catalytic subunit Pol3 and three smaller subunits Cdc1, Cdc27 and Cdm1. Pol3 binds directly to the B-subunit, Cdc1, which in turn binds the C-subunit, Cdc27. Human Pol delta comprises the same four subunits, and the crystal structure was recently reported of a complex of human p50 and the N-terminal domain of p66, the human orthologues of Cdc1 and Cdc27, respectively. RESULTS: To gain insights into the structure and function of Cdc1, random and directed mutagenesis techniques were used to create a collection of thirty alleles encoding mutant Cdc1 proteins. Each allele was tested for function in fission yeast and for binding of the altered protein to Pol3 and Cdc27 using the two-hybrid system. Additionally, the locations of the amino acid changes in each protein were mapped onto the three-dimensional structure of human p50. The results obtained from these studies identify amino acid residues and regions within the Cdc1 protein that are essential for interaction with Pol3 and Cdc27 and for in vivo function. Mutations specifically defective in Pol3-Cdc1 interactions allow the identification of a possible Pol3 binding surface on Cdc1. CONCLUSION: In the absence of a three-dimensional structure of the entire Pol delta complex, the results of this study highlight regions in Cdc1 that are vital for protein function in vivo and provide valuable clues to possible protein-protein interaction surfaces on the Cdc1 protein that will be important targets for further study.

ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii.

DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx. volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non-essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co-opted as a back-up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.

Contrasting effects of Elg1-RFC and Ctf18-RFC inactivation in the absence of fully functional RFC in fission yeast.

Proliferating cell nuclear antigen loading onto DNA by replication factor C (RFC) is a key step in eukaryotic DNA replication and repair processes. In this study, the C-terminal domain (CTD) of the large subunit of fission yeast RFC is shown to be essential for its function in vivo. Cells carrying a temperature-sensitive mutation in the CTD, rfc1-44, arrest with incompletely replicated chromosomes, are sensitive to DNA damaging agents, are synthetically lethal with other DNA replication mutants, and can be suppressed by mutations in rfc5. To assess the contribution of the RFC-like complexes Elg1-RFC and Ctf18-RFC to the viability of rfc1-44, genes encoding the large subunits of these complexes have been deleted and overexpressed. Inactivation of Ctf18-RFC by the deletion of ctf18+, dcc1+ or ctf8+ is lethal in an rfc1-44 background showing that full Ctf18-RFC function is required in the absence of fully functional RFC. In contrast, rfc1-44 elg1Delta cells are viable and overproduction of Elg1 in rfc1-44 is lethal, suggesting that Elg1-RFC plays a negative role when RFC function is inhibited. Consistent with this, the deletion of elg1+ is shown to restore viability to rfc1-44 ctf18Delta cells.

Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase delta subunit Cdc27.

BACKGROUND: DNA polymerases alpha and delta play essential roles in the replication of chromosomal DNA in eukaryotic cells. DNA polymerase alpha (Pol alpha)-primase is required to prime synthesis of the leading strand and each Okazaki fragment on the lagging strand, whereas DNA polymerase delta (Pol delta) is required for the elongation stages of replication, a function it appears capable of performing on both leading and lagging strands, at least in the absence of DNA polymerase epsilon (Pol epsilon). RESULTS: Here it is shown that the catalytic subunit of Pol alpha, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol delta, both in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM), in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function. CONCLUSIONS: A short protein sequence motif (DPIM) has been identified as mediating Pol alpha-Pol delta interactions in fission yeast. Despite being conserved across species, mutational analysis indicates the DPIM does not play an essential role in vivo, suggesting that interaction between the two polymerases is also non-essential.