Timeless–Tipin interactions with MCM and RPA mediate DNA replication stress response

The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless–Tipin complex, a component of the fork protection complex. We used microscopy approaches, including colocalization analysis and proximity ligation assay, to investigate the spatial localization of the complex during ongoing replication in human cells. Taking advantage of the replication stress induction and the ensuing polymerase–helicase uncoupling, we characterized the Timeless–Tipin localization within the replisome. Replication stress was induced using hydroxyurea (HU) and aphidicolin (APH). While HU depletes the substrate for DNA synthesis, APH binds directly inside the catalytic pocket of DNA polymerase and inhibits its activity. Our data revealed that the Timeless–Tipin complex, independent of the stress, remains bound on chromatin upon stress induction and progresses together with the replicative helicase. This is accompanied by the spatial dissociation of the complex from the blocked replication machinery. Additionally, after stress induction, Timeless interaction with RPA, which continuously accumulates on ssDNA, was increased. Taken together, the Timeless–Tipin complex acts as a universal guardian of the mammalian replisome in an unperturbed S-phase progression as well as during replication stress.


SUPPLEMENTARY FIGURES
Confocal z-stack images were split into separate channels.DAPI (blue) channel was then used for nuclear-mask generation.The mask was used for nuclear signal extraction from red and green channels.After subsequent deconvolution of images, performed in 5 iterations using Iterative Deconvolution 3D ImageJ plugin, the colocalization analysis was performed using coloc2 plugin in ImageJ.Scale bar 5 µm.

FigureFigure S3 .
Figure S1.Secondary antibody specificity.(A) Schematic representation of the experimental setup used to test the specificity of secondary antibodies used in the study.(B) Representative images of immunodetection of proteins of interest using indicated antibodies.Selected secondary or primary antibodies were used to evaluate their non-specific binding and background noise they generate.Scale bar: 5 µm.(C) R Pearson correlation measurement between red and green channels corresponding to experimental conditions shown in A and B. For the comparison purposes the R Pearson correlation between mCherry-PCNA and Timeless was plotted.N (R) = 2 cells (A); 2 cells (B); 6 cells (C); 1 cell (D); 6 cells (E); 59 cells (F).
Figure S5.Deconvolution analysis.(A)To test the effect of deconvolution on image quality, selected images were deconvolved over 5, 10, 15 and 20 iterations as indicated.Scale bar 5 µm.(B) Deconvolved images were subsequently used in the colocalization analysis with coloc2 plugin in ImageJ.The graph represents obtained results.

Figure S6 .Figure S8 .
Figure S6.Pipeline for nuclear intensity signal measurement.Multichannel wide-field images were splitted into separate channels.DAPI (blue) channel was then used for nuclear-mask generation.The cells were classified into S and non S-phase cells based on the number of local maxima in the EdU channel.Nuclei with a count of more than 10 local maxima indicated an Sphase cell.Subsequently, the nuclear signal intensity of the proteins was measured.Scale bar 10 µm.

Figure S9 .
Figure S9.Pipeline for PLA signal evaluation.Multichannel confocal images were splitted into separate channels.DAPI (blue) channel was then used for nuclear-mask generation.The cells were classified into S and non S-phase cells based on the EdU foci number (S phase >10 local maxima) .Subsequently, the number of PLA foci was counted in both S and non S-phase cells.Scale bar 10 µm.