Physical and functional interaction of human Tim with DDX11, the Warsaw breakage syndrome DNA helicase
（Warsaw breakage症候群原因遺伝子ヘリカーゼDDX11と複製因子Timとの物理的機能的相互作用）

− この都医学研セミナーは終了しました。 −

講演要旨

Tim, Tipin and Claspin are mediators of ATR-dependent Chk1 activation in the intra-S phase checkpoint response in metazoans. These factors have been found to also play a critical role in ensuring smooth progression of DNA replication forks in unperturbed conditions and to contribute to the sister chromatid cohesion (SCC) process by a molecular mechanism that has not yet been clarified. Here we present evidence that human Tim establishes a direct physical and functional interaction with the cohesion establishment factor, DDX11 (also known as ChlR1). DDX11 is a super-family 2 (SF2) ATP-dependent DEAH-box DNA helicase, which shares sequence similarity to the Fe-S cluster-containing proteins FANCJ, XPD, RTEL1 within SF2. All these human DNA helicases are implicated in rare genetic syndromes and cancer onset. Homozygous mutations of the DDX11-encoding gene are responsible for a cohesinopathy-related rare disease named Warsaw breakage syndrome (WABS). It was reported that down-regulation of DDX11 in various cellular systems causes SCC defects and increased sensitivity to various DNA-damaging agents. In line with these findings, DDX11 was found to be maximally active in vitro on substrates that mimic key intermediates of DNA replication/repair/recombination reactions, such as forked duplex, three-stranded D-loop and anti-parallel G-quadruplex DNA molecules. We have found that recombinant human Tim enhances the unwinding activity of DDX11 on forked DNA substrates up to 10-fold; whereas, DDX11 resolution of anti-parallel G-quadruplex DNA structures is increased 3-4-fold in the presence of Tim. In contrast, no effect on DDX11 DNA unwinding activity is observed in the presence of Tipin alone. We demonstrate that Tim stimulates a bona fide DDX11 ATPase motor function, because no effect of Tim on DDX11 is observed in DNA helicase control reactions, when ATP is omitted or substituted with ATP-?-S, a poorly hydrolysable ATP-analog, or when a helicase-dead mutant (K50R) of DDX11 is used instead of the wild type protein. Co-immunoprecipitation experiments and surface plasmon resonance measurements indicate that DDX11 directly interacts with Tim (KD = 4.32 x 10-9 M). Interaction sites between Tim and DDX11 have been mapped by analysis of protein deleted forms and peptide microarrays technology. Replication fork analysis using Tim- or DDX11-depleted HeLa cells demonstrated that a deficiency in Tim reduced DNA tract length 2-fold, whereas DDX11 deficiency showed only a modest effect on tract length. Cells that were depleted of Tim and DDX11 displayed a shortened tract length that was comparable to Tim depletion alone. Our findings are consistent with the proposal that Tim and DDX11 are involved in the same cellular pathway, acting at the crossroad between genome stability and SCC establishment.