Tim/Timeless, a member of the 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 physi- cal and functional interaction with DDX11, a super- family 2 iron-sulfur cluster DNA helicase genetically linked to the chromosomal instability disorder War- saw breakage syndrome. Tim stimulates DDX11 un- winding activity on forked DNA substrates up to 10- fold and on bimolecular anti-parallel G-quadruplex DNA structures and three-stranded D-loop approx- imately 4-5-fold. Electrophoretic mobility shift as- says revealed that Tim enhances DDX11 binding to DNA, suggesting that the observed stimulation de- rives from an improved ability of DDX11 to interact with the nucleic acid substrate. Surface plasmon res- onance 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 chro- matin fraction is considerably increased following hydroxyurea exposure. Overall, our studies provide evidence that Tim and DDX11 physically and func- tionally interact and act in concert to preserve repli- cation fork progression in perturbed conditions.