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. 2019 Nov 19;10(1):231–253. doi: 10.1080/19491034.2019.1688932

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© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Timing of replication of major and minor satellite domains.

(a) Central slices of cells coexpressing fluorescently tagged PCNA, MeCP2, and CENPB (GFP-PCNA, MeCP2-miRFP670, CENPB-DsRed) at the different stages of chromocenter replication: a cell, where replication of minor satellite domains followed replication of major satellite DNA is shown. Chromocenters where CENPB domains colocalized with PCNA are marked with yellow arrows. Scale bar: 5 microns. (b) Central slices of a cell, where replication of most of the minor satellite domains preceded replication of major satellite DNA. Chromocenters where CENPB domains colocalized with PCNA are marked with yellow arrows. Scale bar: 5 microns. (c) Time course of replication of an individual chromocenter consisting of one visible major satellite region and three distinct minor satellite domains, hence, corresponding to three chromosomes. Upper panel shows minor satellites that are marked via CENPB-DsRed binding (shown in red) and MeCP2 marked major satellite regions (shown in grayscale). Bottom panel shows an overlay of PCNA signal (grayscale) and three minor satellite regions marked by red circles. The size of each magnified image is about 3 µm, the Z-spacing between the individual slices is 0.5 µm. Yellow arrows mark when PCNA and CENPB signals of the three minor satellite domains in the chromocenter become partially overlaid indicative of ongoing replication at the corresponding minor satellite domain. While the replication of the major satellite is ongoing at the beginning of the observation (0 min), the three minor satellites show specific colocalization later, at 65 min (1), and at 170–260 min (2,3), indicating that replication of minor satellites takes place after or toward the end of the replication of the major satellite portion of the chromocenter. The full time lapse of the whole cell with the chromocenter framed as well as full Z-stacks are presented in Movie 2. (d) Schematic summary of the observed temporal patterns of replication of major and minor satellite components of chromocenters and suggested model for replication dynamics of the centromere and pericentromeric heterochromatin.