DNA replication is highly resilient and persistent under the challenge of mild replication stress

Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild replication stress (RS). Contrary to this proposal, we find that cells exposed to mild RS in fact maintain continued DNA replication throughout G2 and during G2-M transition in RAD51- and RAD52-dependent manners. Persistent DNA synthesis is necessary to resolve replication intermediates accumulated in G2 and disengage an ATR-imposed block to mitotic entry. Because of its continual nature, DNA synthesis at very late replication sites can overlap with chromosome condensation, generating the phenomenon of mitotic DNA synthesis. Unexpectedly, we find that the commonly used CDK1 inhibitor RO3306 interferes with replication to preclude detection of G2 DNA synthesis, leading to the impression of a mitosis-driven response. Our study reveals the importance of persistent DNA replication and checkpoint control to lessen the risk for severe genome under-replication under mild RS

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA + ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability. </p

Epigenetic regulators and asymmetric cell division: distinct functions of Haspin during self-renewal and differentiation of embryonic stem cells

The atypical protein kinase Haspin is responsible for mitotic phosphorylation of Histone H3 at threonine-3 (H3T3ph). During mitosis, H3T3ph localizes in the inner centromere and it is thought to provide a docking site for the Chromosomal Passenger Complex (CPC). Although CPC targeting to the centromere is critical for the control of chromosome congression and sister-chromatid segregation, Haspin-knockout mice develop normally and do not exhibit major defects, except for testicular anomalies. To distinguish between essential and non-essential functions of Haspin, we have examined mouse embryonic stem cells lacking or overexpressing this protein. The results show that Haspin is not essential for the population expansion of naïve pluripotent cells. However, the lack of Haspin compromises the in vitro differentiation and its inappropriate expression affects severely the expression of testis-specific genes. According to these data and in combination with observations, we suggest that H3T3ph along with H3K4me3, function as an epigenetic switch that regulates the expression of testis-specific genes during spermiogenesis.Η άτυπη πρωτεϊνική κινάση Haspin είναι υπεύθυνη για τη φωσφορυλίωση της ιστόνης Η3 στη θρεονίνη 3 (H3T3ph). Κατά τη διάρκεια της μίτωσης, η H3T3ph εντοπίζεται στα κεντρομερίδια, παρέχοντας μια θέση πρόσδεσης για το σύμπλοκο CPC (Chromosomal Passenger Complex). Παρότι η σύνδεση του συμπλόκου CPC στο κεντρομερίδιο είναι σημαντική για τη σωστή διάταξη των χρωμοσωμάτων και τον διαχωρισμό των αδελφών χρωματίδων, ποντίκια στα οποία το γονίδιο της Haspin έχει αδρανοποιηθεί (KnockOut-KO) αναπτύσσονται φυσιολογικά χωρίς να παρουσιάζουν σημαντικά ελλείμματα, εκτός από κάποιες ανωμαλίες που παρατηρούνται στους όρχεις. Για να διαχωριστούν οι ζωτικές και μη ζωτικές λειτουργίες της Haspin, εξετάστηκαν εμβρυονικά βλαστικά κύτταρα ποντικού στα οποία έχει απαλειφθεί ή υπερεκφράζεται η συγκεκριμένη πρωτεΐνη. Τα αποτελέσματα δείχνουν ότι η Haspin δεν είναι απαραίτητη για την αυτο-ανανέωση και την έκπτυξη των πολυδύναμων κυττάρων. Παρόλα αυτά, η απουσία της επηρεάζει την in vitro διαφοροποίηση και η αστάθμητη έκφρασή της επηρεάζει σοβαρά την έκφραση ορχεο-ειδικών γονιδίων. Επί τη βάσει αυτών και άλλων συνηγορητικών στοιχείων, προτείνεται ότι η φωσφορυλίωση της θρεονίνης-3 από τη Haspin, μαζί με την τρι-μεθυλίωση της λυσίνης-4 στην ιστόνη Η3 (H3K4me3), λειτουργούν ως ένας επιγενετικός «διακόπτης», που ρυθμίζει την έκφραση αναπτυξιακά σημαντικών γονιδίων κατά τη διάρκεια της σπερμιογένεση

<b>Research data for Centromere protection requires strict mitotic inactivation of the Bloom syndrome helicase complex</b>

This record contains the figures to support the article. Article abstract The BTRR (BLM/TOP3A/RMI1/RMI2) complex resolves various DNA replication and recombination intermediates to suppress genome instability. Alongside PICH, they target mitotic DNA intertwinements, known as ultrafine DNA bridges, facilitating chromosome segregation. Both BLM and PICH undergo transient mitotic hyper-phosphorylation, but the biological significance of this remains elusive. Here, we uncover that during early mitosis, multiple protein kinases act together to strictly constrain the BTRR complex for the protection of centromeres. Mechanistically, CDK1 destabilises the complex and suppresses its association with PICH at the chromatin underneath kinetochores. Inactivating the BLM and TOP3A interaction compromises the UFB-binding complex mitotic functions and can prevent centromere destruction. We further unravel how different clusters of mitotic phosphorylation on BLM affect its interaction with the TOP3A/RMI1/RMI2 subcomplex and illegitimate centromere unwinding. Furthermore, we identify specific phosphorylation sites targeted by the MPS1-PLK1 axis functioning to prevent BLM hyper-activation at centromeres. Notably, unleashing such activity after sister-chromatid cohesion loss facilitates separation of entangled chromosomes. Together, our study defines a centromere protection pathway in human mitotic cells, heavily reliant on a tight spatiotemporal control of the BTRR complex.</p

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability