Amino acids of the Sulfolobus solfataricus mini-chromosome maintenance-like DNA helicase involved in DNA binding/remodeling.

Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. The crystallographic structure of the N-terminal portion (residues 2–286) of the Methanothermobacter thermoautotrophicum MCM protein revealed a dodecameric assembly with two hexameric rings in a head-to-head configuration and a positively charged central channel proposed to encircle DNA molecules. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity. These results have important implications for the understanding of the molecular mechanism of the MCM DNA helicase action

A CDC6-like factor from the archaea Sulfolobus solfataricus promotes binding of the mini-chromosome maintenance complex to DNA

The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes. Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities. These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms

Biochemical characterization of a CDC6-like protein from the crenarchaeon sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function

Take a Break to Repair: A Dip in the World of Double-Strand Break Repair Mechanisms Pointing the Gaze on Archaea

All organisms have evolved many DNA repair pathways to counteract the different types of DNA damages. The detection of DNA damage leads to distinct cellular responses that bring about cell cycle arrest and the induction of DNA repair mechanisms. In particular, DNA double-strand breaks (DSBs) are extremely toxic for cell survival, that is why cells use specific mechanisms of DNA repair in order to maintain genome stability. The choice among the repair pathways is mainly linked to the cell cycle phases. Indeed, if it occurs in an inappropriate cellular context, it may cause genome rearrangements, giving rise to many types of human diseases, from developmental disorders to cancer. Here, we analyze the most recent remarks about the main pathways of DSB repair with the focus on homologous recombination. A thorough knowledge in DNA repair mechanisms is pivotal for identifying the most accurate treatments in human diseases

NurA Is Endowed with Endo- and Exonuclease Activities that Are Modulated by HerA: New Insight into Their Role in DNA-End Processing.

The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities are not strictly dependent on the presence of HerA, require divalent ions (preferably Mn2+), and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue, the endonuclease activity on circular double-stranded DNA is HerA-independent and is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing

The DNA primase of Sulfolobus solfataricus is activated by substrates containing a thymine-rich bubble and has a 3′-terminal nucleotidyl-transferase activity

DNA primases are responsible for the synthesis of the short RNA primers that are used by the replicative DNA polymerases to initiate DNA synthesis on the leading- and lagging-strand at the replication fork. In this study, we report the purification and biochemical characterization of a DNA primase (Sso DNA primase) from the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The Sso DNA primase is a heterodimer composed of two subunits of 36 kDa (small subunit) and 38 kDa (large subunit), which show sequence similarity to the eukaryotic DNA primase p60 and p50 subunits, respectively. The two polypeptides were co-expressed in Escherichia coli and purified as a heterodimeric complex, with a Stokes radius of about 39.2 Å and a 1:1 stoichiometric ratio among its subunits. The Sso DNA primase utilizes poly-pyrimidine single-stranded DNA templates with low efficiency for de novo synthesis of RNA primers, whereas its synthetic function is specifically activated by thymine-containing synthetic bubble structures that mimic early replication intermediates. Interestingly, the Sso DNA primase complex is endowed with a terminal nucleotidyl-tranferase activity, being able to incorporate nucleotides at the 3′ end of synthetic oligonucleotides in a non-templated manner

Physical and functional interaction between the mini-chromosome maintenance-like DNA helicase and the single-stranded DNA binding protein from the crenarchaeon Sulfolobus solfataricus.

Mini-chromosome Maintenance (MCM) proteins play an essential role in both initiation and elongation phases of DNA replication in Eukarya. Genes encoding MCM homologs are present also in the genomic sequence of Archaea and the MCM-like protein from the euryarchaeon Methanobacterium thermoautotrophicum (Mth MCM) was shown to possess a robust ATP-dependent 3'-5' DNA helicase activity in vitro. Herein, we report the first biochemical characterization of a MCM homolog from a crenarchaeon, the thermoacidophile Sulfolobus solfataricus (Sso MCM). Gel filtration and glycerol gradient centrifugation experiments indicate that the Sso MCM forms single hexamers (470 kDa) in solution, whereas the Mth MCM assembles into double hexamers. The Sso MCM has NTPase and DNA helicase activity, which preferentially acts on DNA duplexes containing a 5'-tail and is stimulated by the single-stranded DNA binding protein from S. solfataricus (Sso SSB). In support of this functional interaction, we demonstrated by immunological methods that the Sso MCM and SSB form protein.protein complexes. These findings provide the first in vitro biochemical evidence of a physical/functional interaction between a MCM complex and another replication factor and suggest that the two proteins may function together in vivo in important DNA metabolic pathways

Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus

Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80°C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3–4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP

Synergic and opposing activities of thermophilic RecQ-like helicase and topoisomerase 3 proteins in Holliday junction processing and replication fork stabilization

RecQ family helicases and topoisomerase 3 enzymes form evolutionary conserved complexes that play essential functions in DNA replication, recombination, and repair, and in vitro, show coordinate activities on model recombination and replication intermediates. Malfunctioning of these complexes in humans is associated with genomic instability and cancer-prone syndromes. Although both RecQ-like and topoisomerase 3 enzymes are present in archaea, only a few of them have been studied, and no information about their functional interaction is available. We tested the combined activities of the RecQ-like helicase, Hel112, and the topoisomerase 3, SsTop3, from the thermophilic archaeon Sulfolobus solfataricus. Hel112 showed coordinate DNA unwinding and annealing activities, a feature shared by eukaryotic RecQ homologs, which resulted in processing of synthetic Holliday junctions and stabilization of model replication forks. SsTop3 catalyzed DNA relaxation and annealing. When assayed in combination, SsTop3 inhibited the Hel112 helicase activity on Holliday junctions and stimulated formation and stabilization of such structures. In contrast, Hel112 did not affect the SsTop3 DNA relaxation activity. RecQ-topoisomerase 3 complexes show structural similarity with the thermophile-specific enzyme reverse gyrase, which catalyzes positive supercoiling of DNA and was suggested to play a role in genome stability at high temperature. Despite such similarity and the high temperature of reaction, the SsTop3-Hel112 complex does not induce positive supercoiling and is thus likely to play different roles. We propose that the interplay between Hel112 and SsTop3 might regulate the equilibrium between recombination and anti-recombination activities at replication forks