How unfinished business from S-phase affects mitosis and beyond

Abstract

The eukaryotic cell cycle is conventionally viewed as comprising several discrete steps, each of which must be completed before the next one is initiated. However, emerging evidence suggests that incompletely replicated, or unresolved, chromosomes from S-phase can persist into mitosis, where they present a potential threat to the faithful segregation of sister chromatids. In this review, we provide an overview of the different classes of loci where this 'unfinished S-phase business' can lead to a variety of cytogenetically distinct DNA structures throughout the various steps of mitosis. Furthermore, we discuss the potential ways in which cells might not only tolerate this inevitable aspect of chromosome biology, but also exploit it to assist in the maintenance of genome stability.

Figure 1

DNA replication problems. Schematic examples of obstacles that can hinder DNA replication forks. In some cases, these obstacles can interfere with the timely completion of DNA replication in S-phase, leading to subsequent problems in mitosis. The obstacles depicted from top to bottom are: (i) A DNA adduct (red star). (ii) A DNA repair intermediate, in this case a single-strand DNA break. (iii) A conflict between DNA replication and transcription (RNA polymerase II is depicted in blue and the transcript is shown in orange). (iv) A DNA-bound protein (shown as a green oval). (v) An intra-molecular ssDNA secondary structure in the leading strand, such as G-quadruplex DNA. (vi) Two converging replication forks with ensuing DNA topological stress that would form between them.

Figure 2

Examples of DNA structures arising in S-phase that may cause problems for chromosome segregation in mitosis unless resolved. (Upper left) A late replication intermediate (i), which escapes cleavage by MUS81-EME1 and contains a short stretch of unreplicated ssDNA, can be unwound by the Bloom’s complex (depicted as BTRR). As a result of this, the chromosomes may contain regions of ssDNA that persist into mitosis. Fully replicated chromosomes contain dsDNA catenanes (ii), forming at replication termination sites. TopoIIα is the main enzyme that can decatenated these DNA entanglements, to produce two fully replicated dsDNA duplexes. In principle, the concerted action of the BTRR complex may also contribute to their decatenation. (Right) A double Holliday junction (iii) arises as an intermediate of homologous recombination repair of DNA gaps and breaks in S-phase. This structure can be resolved by the Bloom’s complex (depicted as BTRR) in a process known as ‘double Holliday junction dissolution’, resulting in non-crossover (NCO) dsDNA products. Alternatively, Holliday junctions can be resolved by structure-specific endonucleases, such as MUS81-EME1 or GEN1. Crossover (CO) or NCO dsDNA products are formed depending on the relative orientation of the cleavage and ligation products. A hemicatenane (iv) can form at sites of converging DNA replication forks or as an intermediate product in the double Holliday junction dissolution reaction. Hemicatenanes are efficiently resolved by the TopoIIIα component of the Bloom’s complex, resulting in NCO dsDNA molecules. (Lower left) An R-loop (v), formed during transcription, comprises an DNA:RNA hybrid with a region of displaced ssDNA. Specific DNA:RNA helicases can disrupt R-loops; the RNA transcripts dissociate and the DNA complementary strands are annealed. Alternatively, RNases may digest the RNA transcript.

Figure 3

Normal versus aberrant mitotic structures in human cells. (A) Representative images of a normal mitosis in human U2OS cells. DNA is stained with DAPI (blue) and microtubules are detected using an anti-alpha-tubulin antibody (red). In prophase (not shown), chromosomes begin to condense and the centrosomes separate and move to opposite poles. In prometaphase (i) the nuclear membrane breaks down; the kinetochores assemble at centromeres and are captured by the microtubules. In metaphase (ii) chromosomes appear to be hyper-condensed; sister chromatids are attached in a bipolar fashion to spindle microtubules and aligned on the metaphase plate. In anaphase (iii) cohesin is removed from centromeres by separase-mediated cleavage, and the sister chromatids are pulled to opposite poles of the spindle. In telophase (iv) the midbody forms and cytokinesis occurs via ingression of a cleavage furrow from the plasma membrane. Following abscission, the chromosomes and nuclear components are repackaged into daughter cell nuclei. Finally, chromosomes de-condense and the nuclear envelope re-forms. (B) Examples of aberrant mitotic structures (i) a micronucleus, revealed by DAPI staining (blue) and shown to contain PICH foci (red). (ii) A broken common fragile site, marked by the presence of FANCD2 (red). The green ‘sister foci’ represent centromeres defined by Hec1 kinetochore protein. (iii) An ultrafine DNA bridge, stained positive for BLM (red) with FANCD2 foci (green) on both ends. (iv) A bulky DNA bridge, which is positive for BLM (green), PICH (red), and DAPI staining (blue). (v) 53BP1 bodies (green) in G1 cells, which are defined by being negative for cyclin A staining, as indicated in the figure.