Douglas, M.E.
Diffley, J.F.
(2021). Budding yeast Rap1, but not telomeric DNA, is inhibitory for multiple stages of DNA replication in vitro. Nucleic acids research,
Vol.49
(10),
pp. 5671-5683.
show abstract
Abstract
Telomeres are copied and reassembled each cell division cycle through a multistep process called telomere replication. Most telomeric DNA is duplicated semiconservatively during this process, but replication forks frequently pause or stall at telomeres in yeast, mouse and human cells, potentially causing chronic telomere shortening or loss in a single cell cycle. We have investigated the cause of this effect by examining the replication of telomeric templates in vitro. Using a reconstituted assay for eukaryotic DNA replication in which a complete eukaryotic replisome is assembled and activated with purified proteins, we show that budding yeast telomeric DNA is efficiently duplicated in vitro unless the telomere binding protein Rap1 is present. Rap1 acts as a roadblock that prevents replisome progression and leading strand synthesis, but also potently inhibits lagging strand telomere replication behind the fork. Both defects can be mitigated by the Pif1 helicase. Our results suggest that GC-rich sequences do not inhibit DNA replication per se, and that in the absence of accessory factors, telomere binding proteins can inhibit multiple, distinct steps in the replication process..
Capalbo, L.
Bassi, Z.I.
Geymonat, M.
Todesca, S.
Copoiu, L.
Enright, A.J.
Callaini, G.
Riparbelli, M.G.
Yu, L.
Choudhary, J.S.
Ferrero, E.
Wheatley, S.
Douglas, M.E.
Mishima, M.
D’Avino, P.P.
(2019). The midbody interactome reveals unexpected roles for PP1 phosphatases in cytokinesis. Nature communications,
Vol.10
(1).
show abstract
Abstract
The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1β-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins..
Douglas, M.E.
Ali, F.A.
Costa, A.
Diffley, J.F.
(2018). The mechanism of eukaryotic CMG helicase activation. Nature,
Vol.555
(7695),
pp. 265-268.
Goswami, P.
Abid Ali, F.
Douglas, M.E.
Locke, J.
Purkiss, A.
Janska, A.
Eickhoff, P.
Early, A.
Nans, A.
Cheung, A.M.
Diffley, J.F.
Costa, A.
(2018). Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome. Nature communications,
Vol.9
(1).
Frigola, J.
He, J.
Kinkelin, K.
Pye, V.E.
Renault, L.
Douglas, M.E.
Remus, D.
Cherepanov, P.
Costa, A.
Diffley, J.F.
(2017). Cdt1 stabilizes an open MCM ring for helicase loading. Nature communications,
Vol.8
(1).
Abid Ali, F.
Douglas, M.E.
Locke, J.
Pye, V.E.
Nans, A.
Diffley, J.F.
Costa, A.
(2017). Cryo-EM structure of a licensed DNA replication origin. Nature communications,
Vol.8
(1).
Douglas, M.E.
Diffley, J.F.
(2016). Recruitment of Mcm10 to Sites of Replication Initiation Requires Direct Binding to the Minichromosome Maintenance (MCM) Complex. Journal of biological chemistry,
Vol.291
(11),
pp. 5879-5888.
Douglas, M.E.
Diffley, J.F.
(2012). Replication Timing: The Early Bird Catches the Worm. Current biology,
Vol.22
(3),
pp. R81-R82.
Douglas, M.E.
Davies, T.
Joseph, N.
Mishima, M.
(2010). Aurora B and 14-3-3 Coordinately Regulate Clustering of Centralspindlin during Cytokinesis. Current biology,
Vol.20
(10),
pp. 927-933.
Douglas, M.E.
Mishima, M.
(2010). Still entangled: Assembly of the central spindle by multiple microtubule modulators. Seminars in cell & developmental biology,
Vol.21
(9),
pp. 899-908.