Tim/Timeless, a member of the replication fork protection complex, operates with the Warsaw breakage syndrome DNA helicase DDX11 in the same fork recovery pathway.
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.
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via Pietro Castellino, 111. 80131 - Napoli, Italy.
Federica
F
Calì
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, NIH Biomedical Research Center, 251 Bayview Blvd, Baltimore, MD 21224 USA.
Sanjay Kumar
SK
Bharti
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via Pietro Castellino, 111. 80131 - Napoli, Italy.
Roberta
R
Di Perna
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, NIH Biomedical Research Center, 251 Bayview Blvd, Baltimore, MD 21224 USA.
Robert M
RM
Brosh
Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via Pietro Castellino, 111. 80131 - Napoli, Italy fm.pisani@ibp.cnr.it.
Francesca M
FM
Pisani
D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics; D054856:G-Quadruplexes; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; C509769:TIMELESS protein, human; D004247:DNA; D004265:DNA Helicases; C101830:DDX11 protein, human; D053487:DEAD-box RNA Helicases; D006918:Hydroxyurea