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Review
. 2021 Jun 9;8(6):201932.
doi: 10.1098/rsos.201932.

Mind the replication gap

Camelia Mocanu  1 , Kok-Lung Chan  1
Affiliations
Review

Mind the replication gap

Camelia Mocanu et al. R Soc Open Sci. .

Abstract

Unlike bacteria, mammalian cells need to complete DNA replication before segregating their chromosomes for the maintenance of genome integrity. Thus, cells have evolved efficient pathways to restore stalled and/or collapsed replication forks during S-phase, and when necessary, also to delay cell cycle progression to ensure replication completion. However, strong evidence shows that cells can proceed to mitosis with incompletely replicated DNA when under mild replication stress (RS) conditions. Consequently, the incompletely replicated genomic gaps form, predominantly at common fragile site regions, where the converging fork-like DNA structures accumulate. These branched structures pose a severe threat to the faithful disjunction of chromosomes as they physically interlink the partially duplicated sister chromatids. In this review, we provide an overview discussing how cells respond and deal with the under-replicated DNA structures that escape from the S/G2 surveillance system. We also focus on recent research of a mitotic break-induced replication pathway (also known as mitotic DNA repair synthesis), which has been proposed to operate during prophase in an attempt to finish DNA synthesis at the under-replicated genomic regions. Finally, we discuss recent data on how mild RS may cause chromosome instability and mutations that accelerate cancer genome evolution.

Keywords: break-induced replication; chromosome instability; common fragile sites; mitotic DNA repair synthesis; replication stress; ultrafine DNA bridges.

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Figures

Figure 1.
Figure 1.
ATR coordinates S-phase progression and G2/mitotic entry. (a) Transition between different cell cycle phases is dependent on the activity of cyclins and cyclin-dependent kinase complexes. CDK2 associates with cyclin E during G1 and early S-phase, together with Dbf4-dependent kinase (DDK), Cdc7, they ensure the assembly of pre-replication complexes, which primes the chromatin regions for DNA replication initiation. The CDK2-cyclin A complex is responsible for initiation and completion of DNA synthesis. During late S-phase, CDK1 is also activated by binding to cyclin A, and later by cyclin B, which drives transcription signals to activate the mitotic gene network. During unperturbed conditions, the pre-replication complex (pre-RC) is assembled on chromatin. In G1/S, Cdc6 binds the pre-RC, leading to the recruitment of factors required for licensing of DNA replication (not shown). The Cdc7/Dbf4 kinase complex phosphorylates the pre-RC, allowing for DNA replication to start. CDK2-cyclin E/A complex represses Cdc6 to re-associate with the origins during S-phase, which hence prevents DNA re-replication. Furthermore, this kinase complex also promotes ATR-Chk1, CDK1 and PLK1 activity. As the ATR-Chk1 signalling pathway represses CDK1 activity, a feedback loop is created which ensures that G2 phase commences until S-phase has been nearly completed (i). Under RS conditions, S-phase length is extended as a consequence of prolonged ATR-Chk1 signalling activation (ii). Loss of Cdc6 and Cdc7 functions leads to a failure of DNA replication licensing and the subsequent replication. This diminishes the activation of ATR pathway, leading to an accelerated progression of G2/M phases (iii). (b) ATR couples S-phase progression with G2/mitotic entry by inhibiting both CDK2 and CDK1 activity (left). This function is critical during replication stress (RS) conditions because cells must ensure full duplication and the genome prior to mitotic entry (right). During RS, increased ATR activity leads to inhibition of CDK2, which in turn downregulates global origin firing, prolonging the S-phase. Furthermore, ATR also delays the activation of the FOXM1-mediated mitotic gene network expression by inhibiting the CDK1 activity. Additionally, ATR prevents stalled fork collapse by regulating local origin firing pattern, although the mechanism is less well understood.
Figure 2.
Figure 2.
The replication stress response. (a) During unperturbed S-phase, the CMG complex unwinds the DNA duplex for DNA synthesis mediated by two distinct DNA polymerases, Polδ and Polε, on the lagging and leading strand, respectively. PCNA acts as a processivity factor of both DNA polymerases. Following replication stress, the MCM helicase may uncouple from the DNA polymerase, leading to the generation of a long tract of ssDNA. RPA binds and stabilizes the ssDNA structure. Given the discontinuous synthesis of the lagging strand by Okazaki fragment formation, stalling Polδ may only lead to short stretches or gaps of ssDNA. On the other hand, uncoupling CMG from the Polε DNA polymerase on the lagging strand can generate a long region of ssDNA. The accumulation of bound RPA will then lead to the activation of ATR-CHK1 checkpoint signalling. (b) Upon the coating of the ssDNA by RPA at the stalled fork, ATR is recruited through its partner, ATRIP. To facilitate ATR signalling, two addition proteins, ETAA1 and TopBP1 are required. ETAA1 is recruited to the stalled fork through its interaction with RPA. By contrast, the activation of TopBP1 requires the presence of the Rad9-Rad1-Hus1 (9-1-1) clamp. Loading of the 9-1-1 clamp by the Rad17 clamp loader (not shown) requires the presence of a 5′ ssDNA-dsDNA junction, which is not normally present at a stalled leading strand. PrimPol can be recruited at the stalled fork and initiate primer synthesis, which creates a 5′ ssDNA-dsDNA junction. Successful activation of TopBP1 would lead to the subsequent activation of ATR. In turn, ATR phosphorylates its downstream partner, Chk1, leading to a cascade of checkpoint signalling and stalled fork repairing events. (c) Fork uncoupling also leads to fork remodelling processes. RAD51 replaces RPA on the ssDNA region in a BRCA2-dependent manner to stabilize the stalled fork. ZRANB3 is recruited following DNA-damage-induced ubiquitination of PCNA while SMARCAL1 is recruited via its direct interaction with RPA. HLTF binds the 3′ –OH group on the stalled leading strand. The binding of these fork remodelling factors mediates fork reversal, leading to the formation of a chicken foot structure via a yet unclear mechanism. The regressed arm of the fork becomes a single-ended double-stranded break and is prone to cleavage by nucleases (e.g. CtlP, MRE11, DNA2) but is protected by RAD51 in an enzymatic activity-independent manner. (d) Possible pathways to restart stalled forks. The regressed fork can be stabilized and protected from degradation until it is rescued by a nearby travelling converging fork (1). RAD51 can use its strange-exchange enzymatic activity to promote formation of a D-loop that promotes the re-initiation of replication, especially at regions of highly repetitive sequences (2). Regressed fork can also be restarted by the branch-migrating activity of RECQ1 to restore a three-way replication fork structure (3). Lastly, WRN2-DNA2 controlled resection can also promote fork restart, presumably through the recruitment of other branch migration factors, although the mechanism is not clear (4).
Figure 3.
Figure 3.
The MiDAS/break-induced mitotic DNA repair synthesis model. (a) Under mild replication stress (RS), cells enter mitosis with stalled forks at common fragile sites (CFSs) predominantly. The uncoupling of polymerases causes ssDNA accumulation, leading to RPA and RAD51 filament formation and the binding of FANCD2/I dimer, presumably to protect and stabilize the stalled forks. Upon mitotic entry, CDK1 phosphorylates multiple MiDAS promoting factors including RECQL5 helicase, EME1 (a partner of MUS81 endonuclease) and TRAIP. (b) TRAIP poly-ubiquitinates MCM helicase, driving replisome disassembly. In parallel, the activated MUS81-EME1 is recruited probably via the scaffold platform of SLX4. The phosphorylated RECQL5 removes the RAD51 filaments from the leading strand. Furthermore, RECQL5 can also physically associate with MUS81 that promotes its endonuclease activity. (c) Consequently, MUS81-EME1 probably cleaves the leading strand of the stalled fork and generates a single-ended DSB. (d) To generate a 3′ ssDNA overhang for homologous strand invasion, the cleaved arm is probably undergoing a 5′ to 3′ resection by an exonuclease(s). (e) RAD52 mediates the strand annealing step followed by the formation of D-loop. A POLD3-containing polymerase then resynthesizes DNA at the incompletely replicated regions ahead. Helicases like PIF1 or MCM may need to facilitate the DNA synthesis progression. (i) The D-loop can migrate with the POLD3-containing polymerase that may hit a neighbouring stalled fork. As a result, a Holliday junction (HJ)-like structure is formed. The gap of the nascent synthesized strand may be filled (the purple dotted line) during D-loop migration or after the HJ resolution, resulting in a conservative DNA synthesis. (ii) If the D-loop is resolved immediately after the stand invasion, it may restore a three-way replication fork structure for DNA resynthesis in a semi-conservative manner. (iii) Alternatively, the stalled converging forks can both initiate BIR, leading to the formation of a double D-loop. DNA resynthesis between the D-loops will fill up the under-replication gap region and generate dsDNA catenane. The single or double D-loop may then be cleaved by MUS81-EME1 and/or other structural-specific endonucleases to resolve sister-DNA entanglements.
Figure 4.
Figure 4.
Mild replication stress leads to chromosome segregation defects that increase genome instability. (a) Cells can commit mitosis despite the presence of under-replicated DNA structures under mild replication stress conditions. This leads to the interlinkage of sister chromatids. FANCD2/I dimer and MUS81-EME1-SLX4 complex are recruited to these structures, presumably to protect or resolve the DNA linkages. This has been suggested to cause the cytogenetic breaks and/or gaps on the mitosis chromosomes. The stalled fork breakage due to the MUS81-EME1 cleavage was proposed to initiate MiDAS/BIR reaction. If MiDAS is completed, it will convert the stalled fork structure to recombination structures such as D-loop or HJ-like molecules. They may be resolved during prophase/prometaphase or, otherwise, can generate ultrafine DNA bridges in anaphase cells, which may be resolved by the PICH/BLM complex. If MiDAS is defective or incomplete, sister chromatids remain intertwined by the replication intermediates that again can form UFBs and/or bulky chromatin bridges during anaphase. The failure or inappropriate resolution of the replication intermediates can lead to chromosome non-disjunction and/or chromatin damage, resulting in micronuclei formation or 53BP1 nuclear bodies in the newborn G1 daughter cells. (b) Mild RS has also been shown to increase aneuploidy as it can alter microtubule (MT) spindle dynamics and induces premature centriole disengagement. This presumably promotes erroneous merotelic-kinetochore attachments in metaphase, which can lead to lagging chromosome formation during anaphase. Alternatively, premature centriole disengagement can cause multi-polar spindles formation and chromosome mis-segregation. It was hypothesized that the centrosomes have accelerated the maturation cycle as a result of prolonged S/G2 delay induced by replication stress. Under these conditions, it increases the chances of whole chromosome mis-segregation.
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