Restarted replication forks are error-prone and cause CAG repeat expansions and contractions.

Disease-associated trinucleotide repeats form secondary DNA structures that interfere with replication and repair. Replication has been implicated as a mechanism that can cause repeat expansions and contractions. However, because structure-forming repeats are also replication barriers, it has been unclear whether the instability occurs due to slippage during normal replication progression through the repeat, slippage or misalignment at a replication stall caused by the repeat, or during subsequent replication of the repeat by a restarted fork that has altered properties. In this study, we have specifically addressed the fidelity of a restarted fork as it replicates through a CAG/CTG repeat tract and its effect on repeat instability. To do this, we used a well-characterized site-specific replication fork barrier (RFB) system in fission yeast that creates an inducible and highly efficient stall that is known to restart by recombination-dependent replication (RDR), in combination with long CAG repeat tracts inserted at various distances and orientations with respect to the RFB. We find that replication by the restarted fork exhibits low fidelity through repeat sequences placed 2-7 kb from the RFB, exhibiting elevated levels of Rad52- and Rad8ScRad5/HsHLTF-dependent instability. CAG expansions and contractions are not elevated to the same degree when the tract is just in front or behind the barrier, suggesting that the long-traveling Polδ-Polδ restarted fork, rather than fork reversal or initial D-loop synthesis through the repeat during stalling and restart, is the greatest source of repeat instability. The switch in replication direction that occurs due to replication from a converging fork while the stalled fork is held at the barrier is also a significant contributor to the repeat instability profile. Our results shed light on a long-standing question of how fork stalling and RDR contribute to expansions and contractions of structure-forming trinucleotide repeats, and reveal that tolerance to replication stress by fork restart comes at the cost of increased instability of repetitive sequences.

The DNA Replication Checkpoint Targets the Kinetochore for Relocation of Collapsed Forks to the Nuclear Periphery.

Hairpin forming expanded CAG/CTG repeats pose significant challenges to DNA replication which can lead to replication fork collapse. Long CAG/CTG repeat tracts relocate to the nuclear pore complex to maintain their integrity. Forks impeded by DNA structures are known to activate the DNA damage checkpoint, thus we asked whether checkpoint proteins play a role in relocation of collapsed forks to the nuclear periphery in S. cerevisiae . We show that relocation of a (CAG/CTG) 130 tract is dependent on activation of the Mrc1/Rad53 replication checkpoint. Further, checkpoint-mediated phosphorylation of the kinetochore protein Cep3 is required for relocation, implicating detachment of the centromere from the spindle pole body. Activation of this pathway leads to DNA damage-induced microtubule recruitment to the repeat. These data suggest a role for the DNA replication checkpoint in facilitating movement of collapsed replication forks to the nuclear periphery by centromere release and microtubule-directed motion. Highlights: The DNA replication checkpoint initiates relocation of a structure-forming CAG repeat tract to the nuclear pore complex (NPC)The importance of Mrc1 (hClaspin) implicates fork uncoupling as the initial checkpoint signalPhosphorylation of the Cep3 kinetochore protein by Dun1 kinase allows for centromere release, which is critical for collapsed fork repositioningDamage-inducible nuclear microtubules (DIMs) colocalize with the repeat locus and are required for relocation to the NPCEstablishes a new role for the DNA replication and DNA damage checkpoint response to trigger repositioning of collapsed forks within the nucleus.

A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast.

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.

Location, Location, Location: The Role of Nuclear Positioning in the Repair of Collapsed Forks and Protection of Genome Stability.

Components of the nuclear pore complex (NPC) have been shown to play a crucial role in protecting against replication stress, and recovery from some types of stalled or collapsed replication forks requires movement of the DNA to the NPC in order to maintain genome stability. The role that nuclear positioning has on DNA repair has been investigated in several systems that inhibit normal replication. These include structure forming sequences (expanded CAG repeats), protein mediated stalls (replication fork barriers (RFBs)), stalls within the telomere sequence, and the use of drugs known to stall or collapse replication forks (HU + MMS or aphidicolin). Recently, the mechanism of relocation for collapsed replication forks to the NPC has been elucidated. Here, we will review the types of replication stress that relocate to the NPC, the current models for the mechanism of relocation, and the currently known protective effects of this movement.

Relocation of Collapsed Forks to the Nuclear Pore Complex Depends on Sumoylation of DNA Repair Proteins and Permits Rad51 Association.

Expanded CAG repeats form stem-loop secondary structures that lead to fork stalling and collapse. Previous work has shown that these collapsed forks relocalize to nuclear pore complexes (NPCs) in late S phase in a manner dependent on replication, the nucleoporin Nup84, and the Slx5 protein, which prevents repeat fragility and instability. Here, we show that binding of the Smc5/6 complex to the collapsed fork triggers Mms21-dependent sumoylation of fork-associated DNA repair proteins, and that RPA, Rad52, and Rad59 are the key sumoylation targets that mediate relocation. The SUMO interacting motifs of Slx5 target collapsed forks to the NPC. Notably, Rad51 foci only co-localize with the repeat after it is anchored to the nuclear periphery and Rad51 exclusion from the early collapsed fork is dependent on RPA sumoylation. This pathway may provide a mechanism to constrain recombination at stalled or collapsed forks until it is required for fork restart.

The nuclear pore complex prevents sister chromatid recombination during replicative senescence.

The Nuclear Pore Complex (NPC) has emerged as an important hub for processing various types of DNA damage. Here, we uncover that fusing a DNA binding domain to the NPC basket protein Nup1 reduces telomere relocalization to nuclear pores early after telomerase inactivation. This Nup1 modification also impairs the relocalization to the NPC of expanded CAG/CTG triplet repeats. Strikingly, telomerase negative cells bypass senescence when expressing this Nup1 modification by maintaining a minimal telomere length compatible with proliferation through rampant unequal exchanges between sister chromatids. We further report that a Nup1 mutant lacking 36 C-terminal residues recapitulates the phenotypes of the Nup1-LexA fusion indicating a direct role of Nup1 in the relocation of stalled forks to NPCs and restriction of error-prone recombination between repeated sequences. Our results reveal a new mode of telomere maintenance that could shed light on how 20% of cancer cells are maintained without telomerase or ALT.

Budding Yeast BFA1 Has Multiple Positive Roles in Directing Late Mitotic Events.

The proper regulation of cell cycle transitions is paramount to the maintenance of cellular genome integrity. In Saccharomyces cerevisiae, the mitotic exit network (MEN) is a Ras-like signaling cascade that effects the transition from M phase to G1 during the cell division cycle in budding yeast. MEN activation is tightly regulated. It occurs during anaphase and is coupled to mitotic spindle position by the spindle position checkpoint (SPoC). Bfa1 is a key component of the SPoC and functions as part of a two-component GAP complex along with Bub2 The GAP activity of Bfa1-Bub2 keeps the MEN GTPase Tem1 inactive in cells with mispositioned spindles, thereby preventing inappropriate mitotic exit and preserving genome integrity. Interestingly, a GAP-independent role for Bfa1 in mitotic exit regulation has been previously identified. However the nature of this Bub2-independent role and its biological significance are not understood. Here we show that Bfa1 also activates the MEN by promoting the localization of Tem1 primarily to the daughter spindle pole body (dSPB). We demonstrate that the overexpression of BFA1 is lethal due to defects in Tem1 localization, which is required for its activity. In addition, our studies demonstrate a Tem1-independent role for Bfa1 in promoting proper cytokinesis. Cells lacking TEM1, in which the essential mitotic exit function is bypassed, exhibit cytokinesis defects. These defects are suppressed by the overexpression of BFA1 We conclude that Bfa1 functions to both inhibit and activate late mitotic events.

LTE1 promotes exit from mitosis by multiple mechanisms.

In budding yeast, alignment of the anaphase spindle along the mother-bud axis is crucial for maintaining genome integrity. If the anaphase spindle becomes misaligned in the mother cell compartment, cells arrest in anaphase because the mitotic exit network (MEN), an essential Ras-like GTPase signaling cascade, is inhibited by the spindle position checkpoint (SPoC). Distinct localization patterns of MEN and SPoC components mediate MEN inhibition. Most components of the MEN localize to spindle pole bodies. If the spindle becomes mispositioned in the mother cell compartment, cells arrest in anaphase due to inhibition of the MEN by the mother cell-restricted SPoC kinase Kin4. Here we show that a bud-localized activating signal is necessary for full MEN activation. We identify Lte1 as this signal and show that Lte1 activates the MEN in at least two ways. It inhibits small amounts of Kin4 that are present in the bud via its central domain. An additional MEN-activating function of Lte1 is mediated by its N- and C-terminal GEF domains, which, we propose, directly activate the MEN GTPase Tem1. We conclude that control of the MEN by spindle position is exerted by both negative and positive regulatory elements that control the pathway's GTPase activity.