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

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.

A Long Noncoding RNA Regulates Sister Chromatid Cohesion

“…This analysis indicated that most of the genes co-expressed with CONCR (correlation value > 0.45) encode for proteins known to be involved in DNA replication and chromosome maintenance (Figures S2F and S2G and Table S4), including DDX11 and several of its interacting partners, such as TIMELESS (Calì et al, 2016), ESPL1, and components of the minichromosome maintenance complex (MCM) (Figure S2H). This, together with the known function of DDX11 in sister chromatid cohesion, prompted us to investigate the functional role of CONCR in this process.…”

“…DDX11 is a DNA helicase that links the replication of the lagging DNA strand with the establishment of chromosomal cohesion (Bharti et al, 2014), and it is especially required in order to solve stalled replication forks (Calì et al, 2016). Although we cannot exclude that CONCR has additional activities, our results show that CONCR mainly functions at this level.…”

Timeless couples G quadruplex detection with processing by DDX11 during DNA replication

“…Additionally, co-immunoprecipitation experiments show that neither region is required for binding the DDX11 helicase ( Supplementary Fig. 5), a known binding partner of Timeless and whose ability to unwind G4s is stimulated by Timeless 36 . These results indicate that the C terminus of the Timeless protein has an important role in G4 replication with the DBD and the PDB both contributing to that function independently of DDX11 recruitment.…”

“…Although Timeless itself does not appear to possess a catalytic activity that would process DNA secondary structures, it interacts with the helicase DDX11 36 . DDX11 (or CHLR1) is a 5'-3' Fe-S helicase and belongs to the same superfamily 2 as FANCJ, RTEL and XPD 3 .…”

RecQ and Fe–S helicases have unique roles in DNA metabolism dictated by their unwinding directionality, substrate specificity, and protein interactions

“…In yeast, sister chromatid cohesion (which involves the ortholog of human ChlR1/DDX11 [83,84]) is coupled with lagging strand processing [103,104] and involves Ctf4 recruitment of Chl1 to the replisome [105], suggesting that DDX11 interactions with replisome-associated proteins [e.g. Flap Endonuclease 1 (FEN-1) [94] and Timeless [106]] are important (Table 3 and Figure 2). T. acidophilum XPD was shown to unwind a forked duplex, but to our knowledge its preference for that substrate compared with other partial duplex DNA molecules was not evaluated [22].…”