Defective disjunction of sister chromatids can arise due to aberrant DNA structures that are mainly detected during anaphase and are known as ultrafine anaphase bridges. These bridges have to be repaired otherwise the bridge persists into cytokinesis and could give rise to chromosomal instability. Ongoing research is focused on understanding how the cell resolves these bridges before cytokinesis by investigating proteins and protein-protein interactions involved in the resolution mechanism. In this review, the focus will be on the known types of UFBs, the causes of UFBs and the proteins that are involved with bridge resolution.
The cell division cycle represents an interesting challenge to every cell. Before the cell has been duplicated, DNA is replicated to create the sister chromatids that are joined at the centromeres. During mitosis, the chromosomes are evenly distributed by the mitotic spindle that binds to the sister kinetochores at the centromeres.1, 2 The cell division cycle can be efficient if segregation is not compromised.1, 3 Otherwise, genome integrity is destroyed, leading to chromosomal instability that is commonly observed in cancer. Nondisjunction of the sister chromatids is strongly related to abnormal DNA structures that are mainly detected during anaphase, known as anaphase bridges. Previously, anaphase bridges were presumed to be a rare occurrence, however, recent evidence has shown that in most mitosis ultrafine anaphase bridges (UFBs) are generated; these are histone-free DNA threads.3 UFBs cannot be detected by using conventional DNA dyes, but instead the proteins associated with the bridges can be visualised by immunofluorescence staining.1-3 Importantly, the UFBs need to be resolved before the cell continues into cytokinesis because genome integrity needs to be preserved. In this review, the focus will be on the different types of UFBs, the causes of UFBs and the proteins involved with bridge resolution.
Currently, five types of UFBs are known of which two types are caused by catenanes. In physiological conditions, UFBs at the centromeres (C-UFBs) are generally found in mitosis and are produced by double-stranded DNA (dsDNA) catenanes that have not been repaired yet (Figure 1).4 In early mitosis, cohesins are removed from the chromosome arms, whereas cohesins at the centromeres remain until anaphase to preserve the catenanes generated by DNA replication.5, 6 These C-UFBs are associated with the centromeric marker, HEC1, at the termini of the bridge.5 Comparatively, dsDNA catenanes are also the reason for UFBs in ribosomal DNA (rDNA).1 Markedly, rDNA does not condense together with the rest of the DNA at the beginning of mitosis. This delayed condensation results into rDNA catenanes that could not be repaired beforehand and subsequently create rDNA UFBs (R-UFBs) that are associated with the rDNA marker, the upstream binding factor.
Furthermore, two other types of UFBs have been described that are caused by replication or recombination intermediates that persist into anaphase. The DNA loci contains chromosomal fragile sites (CFSs), such as FRA16D or FRA3B, where fragile sites UFBs (FS-UFBs) occur that are associated with the Fanconi anaemia (FA) proteins, FANCD2 and FANCI, at the termini of the bridge (Figure 1).7, 8 The CFSs are stretches of DNA where replication is difficult to complete.8 At these sites, the late replication intermediates (LRIs) have to be solved, otherwise the sister chromatids remain connected, resulting into DNA breakage.7-9 In the daughter cells, DNA breakage are mainly repaired by either single-strand annealing or non-homologous end joining, leading to a loss of heterozygosity and gross chromosome rearrangements. Double-stranded breaks can also be repaired via homologous recombination (HR). The first step is to create 3’ single-stranded DNA (ssDNA) tails that can invade into the DNA duplex of the sister chromatid to create a D-loop structure. These D-loop structures are the initiation sites for DNA replication to form the double Holliday junction (DJH) that keeps the sister chromatids linked to each other.10 The unresolved recombination intermediates cause the formation of HR-UFBs that prevents the evenly distribution of the sister chromatids.
Finally, the fifth type of UFBs is characterised by end-to-end fusion of the telomeric regions on the sister chromatids. The telomeres are normally protected by the shelterin protein complex that contains six subunits, including TRF1 and TRF2.11, 12 When TRF1 is not present, then topoisomerase IIα cannot be employed for its decatenating activity, meaning that the telomeric UFBs (T-UFBs) possibly result from persistent DNA catenanes.11 While TRF2 overexpression, stalls the replication fork at the telomeres because the telomeric regions are difficult to replicate similarly to CFs.12 The LRIs result into T-UFBs and if these are not repaired then the telomeric sequences can be lost as has been found in human cancer.
Schematic diagram representing ultrafine anaphase bridges. The C-UFB is marked by Hec1 and is caused by dsDNA catenanes, whereas FS-UFB is marked by FANCD2/FANCI twin foci and is caused by LRIs.vPotential proteins have been discovered that associate with the bridge during anaphase. The main protein is the Plk1-interacting checkpoint helicase (PICH) that belongs to the SNF2 ATPase family, containing a dsDNA translocase activity.13 Without Plk1, PICH can localise to the centromeres where it has a high binding affinity for dsDNA that is stretched due to tension caused by the mitotic spindle.3, 13 Notably, PICH seems to play a role in C-UFBs, FS-UFBs, R-UFBs and HR-UFBs because as a tension sensing protein PICH may be able to recognise all dsDNA structures under tension.
After PICH has settled on the bridge, this protein can recruit other UFB-associated proteins, such as TOP2A and BLM (Figure 2). The aforementioned TOP2A has a dsDNA decatenating activity that is used to repair the catenanes in C-UFBs and R-UFBs and PICH promotes this activity in vitro.1, 6, 14 However, the decatenating activity of TOP2A has not been demonstrated on stretched dsDNA catenanes. Comparable to PICH, the BLM protein seems to appear on C-UFBs, FS-UFBs and HR-UFBs.5, 10, 15, 16 This protein is from the RecQ family DNA helicase and is usually partnered with topoisomerase IIIα (TOP3A) and RMI1/2 to form the BTRR complex; this complex can associate with both catenanes and LRIs.5, 8, 17, 18 In the case of HR-UFBs, BLM resolves the DJHs, and other types of intermediates, by creating hemicatenanes, which are ssDNA catenanes. These hemicatenanes are repaired by the ssDNA decatenating activity of TOP3A, which is activated by both BLM and RMI1.5, 8, 17 Next to the BTRR complex, the SLX1-SLX4, MUS81-EME1 and XPF-ERCC1 (SMX) tri-nuclease and GEN1 endonuclease cleave the recombination intermediates to promote chromosome segregation.
Although, the BTRR complex has the ability to resolve dsDNA structures, this complex can also damage ssDNA present in UFBs by creating nonspecific breakages.18, 19 When the replication fork is stalled due to replication stress then the stretches of ssDNA are normally protected by the recombination proteins Rad51 and Rad52.9, 20 Without Rad51 and Rad52, stretches of ssDNA are created behind the arrested forks and the sister chromatids remain connected. These ssDNA stretches are stabilised by the replication protein A (RPA) to prevent the breakage caused by the BTRR complex and thus the BTRR complex is mostly anchored to PICH at only dsDNA structures.3, 8, 19 However, RPA has also been found on hemicatenanes after DJH resolution by BLM in HR-UFBs and thus the coordinated activity of BLM and RPA has to be further investigated. Additionally, RPA has been found on ssDNA that was generated after resolution of T-UFBs by the cytoplasmic exonuclease TREX1.
In contrast to PICH and the BTRR complex, other proteins have been discovered but their exact role remains to be investigated. Firstly, Rif1 is recruited by PICH to UFBs, where Rif1 interacts with BLM to repair stalled replication forks.2, 21 Surprisingly, Rif1 has been on T-UFBs where it prevents the resolution of these bridges, whereas it stimulates the resolution of the other types of UFBs.22 Secondly, TOPBP1 has been found in a subset of UFBs together with PICH.23 This protein possibly inhibits recombination of the stalled replication forks and in some cases also appears to recruit TOP2A for its decatenating activity.
A model representing the UFB-associated proteins. (1) C-UFBs are decorated with PICH and BLM and repaired by TOP2A. The ssDNA are stabilised by RPA (pink circles). (2) FS-UFBs are marked by the FANCD2/FANCI twin foci on the bridge termini. The bridges are coated by PICH, BLM and RPA. (3) T-UFB are processed by TREX1 to create RPA-coated ssDNA. (4) The recombinatin intermediates persist into anaphase without GEN1 and MUS81. The bridges are coated by PICH. BLM generates ssDNA that are subsequently coated by RPA.
The discovery of UFBs explains in part how complex genomic instability can be in cancer cells. Currently, five types of UFBs are known while the proteins involved in the resolution mechanism are still being discovered. PICH and the BTRR complex seem to appear on most UFBs, but the coordinated activity with other proteins remains unclear. In contrast, RPA is localised to ssDNA, but the resolution mechanism of the ssDNA after RPA interaction is yet to be determined. Additionally, the role of the other proteins has to be further investigated to eventually understand UFB resolution. Future research should focus on the protein-protein interactions and the interaction of the protein with each specific UFB to not only understand how the cell resolves UFBs but also to determine how the cell prevents the formation of aberrant DNA structures to prevent pathological situations.
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