The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 A crystal structure of the N-terminal domain (residues 1-268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed.

Mini-chromosome maintenance (MCM) proteins form ring-like hexameric complexes that are commonly believed to act as the replicative DNA helicase at the eukaryotic/archaeal DNA replication fork. Because of their simplified composition with respect to the eukaryotic counterparts, the archaeal MCM complexes represent a good model system to use in analyzing the structural/functional relationships of these important replication factors. In this study the domain organization of the MCM-like protein from Sulfolobus solfataricus (Sso MCM) has been dissected by trypsin partial proteolysis. Three truncated derivatives of Sso MCM corresponding to protease-resistant domains were produced as soluble recombinant proteins and purified: the N-terminal domain (N-ter, residues 1–268); a fragment comprising the AAA+ and C-terminal domains (AAA+-C-ter, residues 269–686); and the C-terminal domain (C-ter, residues 504–686). All of the purified recombinant proteins behaved as monomers in solution as determined by analytical gel filtration chromatography, suggesting that the polypeptide chain integrity is required for stable oligomerization of Sso MCM. However, the AAA+-C-ter derivative, which includes the AAA+ motor domain and retains ATPase activity, was able to form dimers in solution when ATP was present, as analyzed by size exclusion chromatography and glycerol gradient sedimentation analyses. Interestingly, the AAA+-C-ter protein could displace oligonucleotides annealed to M13 single-stranded DNA although with a reduced efficiency in comparison with the full-sized Sso MCM. The implications of these findings for understanding the DNA helicase mechanism of the MCM complex are discussed.

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.

We present evidence that Tim establishes a physical and functional interaction with DDX11, a super-family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder Warsaw breakage syndrome. Tim stimulates DDX11 unwinding activity on forked DNA substrates up to 10-fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approximately 4–5-fold. Electrophoretic mobility shift assays revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation derives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim. DNA fiber track assays with HeLa cells exposed to hydroxyurea demonstrated that Tim or DDX11 depletion significantly reduced replication fork progression compared to control cells; whereas no additive effect was observed by co-depletion of both proteins. Moreover, Tim and DDX11 are epistatic in promoting efficient resumption of stalled DNA replication forks in hydroxyurea-treated cells. This is consistent with the finding that association of the two endogenous proteins in the cell extract chromatin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and functionally interact and act in concert to preserve replication fork progression in perturbed conditions.

Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication and the progression of the replication fork. With GINS, Cdc45 is an essential cofactor of the Mcm2-7 replicative helicase complex. Despite its importance, no detailed information is available on either the structure or the biochemistry of the protein. Intriguingly, whereas homologues of both GINS and Mcm proteins have been described in Archaea, no counterpart for Cdc45 is known. Herein we report a bioinformatic analysis that shows a weak but significant relationship among eukaryotic Cdc45 proteins and a large family of phosphoesterases that has been described as the DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyze the hydrolysis of phosphodiester bonds via a mechanism involving two Mn(2+) ions. Only a subset of the amino acids that coordinates Mn(2+) is conserved in Cdc45. We report biochemical and structural data on the recombinant human Cdc45 protein, consistent with the proposed DHH family affiliation. Like the RecJ exonucleases, the human Cdc45 protein is able to bind single-stranded, but not double-stranded DNA. Small angle x-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members.

Establishment of sister chromatid cohesion is coupled to DNA replication, but the underlying molecular mechanisms are incompletely understood. DDX11 (also named ChlR1) is a super-family 2 Fe-S cluster-containing DNA helicase implicated in Warsaw breakage syndrome (WABS). Herein, we examined the role of DDX11 in cohesion establishment in human cells. We demonstrated that DDX11 interacts with Timeless, a component of the replication fork-protection complex, through a conserved peptide motif. The DDX11-Timeless interaction is critical for sister chromatid cohesion in interphase and mitosis. Immunofluorescence studies further revealed that cohesin association with chromatin requires DDX11. Finally, we demonstrated that DDX11 localises at nascent DNA by SIRF analysis. Moreover, we found that DDX11 promotes cohesin binding to the DNA replication forks in concert with Timeless and that recombinant purified cohesin interacts with DDX11 in vitro. Collectively, our results establish a critical role for the DDX11-Timeless interaction in coordinating DNA replication with sister chromatid cohesion, and have important implications for understanding the molecular basis of WABS. Chromosomes are DNA molecules that contain the genetic information. During replication, the two sister DNA molecules covered by proteins (sister chromatids) are held together by many copies of a ring-like protein complex named cohesin, in a process called sister-chromatid cohesion. Before a cell divides, the cohesin rings are removed from the two sister chromatids to allow their migration towards the opposite poles of the dividing mother cell. At the end of this process, the two daughter cells have inherited a complete set of chromosomes. Before the next cell division, chromosomes are duplicated with high speed and fidelity. This important task is performed by the DNA replication machinery, a sophisticated apparatus made of several enzymes and proteins. In the present study, we have demonstrated that DDX11 and Timeless, two subunits of the DNA replication machinery, recruit the cohesin rings to promote their stable binding to the newly duplicated chromosomes, that is the establishment of sister-chromatid cohesion. In human cells that were genetically engineered to reduce the level of DDX11, we observed that sister-chromatid cohesion was loosened and association of cohesin to chromosomes was reduced. Our experimental results contribute to our understanding of the molecular mechanisms underlying the functional coupling between DNA replication and sister-chromatid cohesion in human cells.