ABSTRACT
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.
Subject(s)
Cell Cycle Proteins/metabolism , DEAD-box RNA Helicases/metabolism , DNA Helicases/metabolism , DNA Replication , Intracellular Signaling Peptides and Proteins/metabolism , Base Sequence , Cell Cycle Proteins/genetics , DEAD-box RNA Helicases/genetics , DNA/chemistry , DNA/metabolism , DNA Helicases/genetics , DNA Replication/genetics , G-Quadruplexes , HeLa Cells/drug effects , Humans , Hydroxyurea/pharmacology , Intracellular Signaling Peptides and Proteins/genetics , Molecular Sequence Data , Nucleic Acid ConformationABSTRACT
The eukaryotic DNA replication protein Mcm10 (mini-chromosome maintenance 10) associates with chromatin in early S-phase and is required for assembly and function of the replication fork protein machinery. Another essential component of the eukaryotic replication fork is Cdc45 (cell division cycle 45), which is required for both initiation and elongation of DNA replication. In the present study we characterize, for the first time, the physical and functional interactions of human Mcm10 and Cdc45. First we demonstrated that Mcm10 and Cdc45 interact in cell-free extracts. We then analysed the role of each of the Mcm10 domains: N-terminal, internal and C-terminal (NTD, ID and CTD respectively). We have detected a direct physical interaction between CTD and Cdc45 by both in vitro co-immunoprecipitation and surface plasmon resonance experiments. On the other hand, we have found that the interaction of the Mcm10 ID with Cdc45 takes place only in the presence of DNA. Furthermore, we found that the isolated ID and CTD domains are fully functional, retaining DNA-binding capability with a clear preference for bubble and fork structures, and that they both enhance Cdc45 DNA-binding affinity. The results of the present study demonstrate that human Mcm10 and Cdc45 directly interact and establish a mutual co-operation in DNA binding.
Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Models, Molecular , Binding Sites , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell-Free System , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Electrophoretic Mobility Shift Assay , HEK293 Cells , Humans , Immunoprecipitation , Kinetics , Minichromosome Maintenance Proteins , Molecular Docking Simulation , Molecular Weight , Nucleic Acid Conformation , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Conformation , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Surface Plasmon ResonanceABSTRACT
The Tim-Tipin complex plays an important role in the S phase checkpoint and replication fork stability in metazoans, but the molecular mechanism underlying its biological function is poorly understood. Here, we present evidence that the recombinant human Tim-Tipin complex (and Tim alone) markedly enhances the synthetic activity of DNA polymerase ε. In contrast, no significant effect on the synthetic ability of human DNA polymerase α and δ by Tim-Tipin was observed. Surface plasmon resonance measurements and co-immunoprecipitation experiments revealed that recombinant DNA polymerase ε directly interacts with either Tim or Tipin. In addition, the results of DNA band shift assays suggest that the Tim-Tipin complex (or Tim alone) is able to associate with DNA polymerase ε bound to a 40-/80-mer DNA ligand. Our results are discussed in view of the molecular dynamics at the human DNA replication fork.
