POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis.

BRCA2-mutant cells are defective in homologous recombination, making them vulnerable to the inactivation of other pathways for the repair of DNA double-strand breaks (DSBs). This concept can be clinically exploited but is currently limited due to insufficient knowledge about how DSBs are repaired in the absence of BRCA2. We show that DNA polymerase θ (POLθ)-mediated end joining (TMEJ) repairs DSBs arising during the S phase in BRCA2-deficient cells only after the onset of the ensuing mitosis. This process is regulated by RAD52, whose loss causes the premature usage of TMEJ and the formation of chromosomal fusions. Purified RAD52 and BRCA2 proteins both block the DNA polymerase function of POLθ, suggesting a mechanism explaining their synthetic lethal relationships. We propose that the delay of TMEJ until mitosis ensures the conversion of originally one-ended DSBs into two-ended DSBs. Mitotic chromatin condensation might further serve to juxtapose correct break ends and limit chromosomal fusions.

Turning end-joining upside down in mitosis.

How cells deal with DNA breaks during mitosis is not well understood. While canonical non-homologous end-joining predominates in interphase, it is inhibited in mitosis to avoid telomere fusions. DNA polymerase θ mediated end-joining appears to be repressed in interphase, but promotes break repair in mitosis. The nature and induction time of breaks might determine their fate during mitosis.

Analysis of novel missense ATR mutations reveals new splicing defects underlying Seckel syndrome.

Ataxia Telangiectasia and Rad3 related (ATR) is one of the main regulators of the DNA damage response. It coordinates cell cycle checkpoint activation, replication fork stability, restart and origin firing to maintain genome integrity. Mutations of the ATR gene have been reported in Seckel patients, who suffer from a rare genetic disease characterized by severe microcephaly and growth retardation. Here, we report the case of a Seckel patient with compound heterozygous mutations in ATR. One allele has an intronic mutation affecting splicing of neighboring exons, the other an exonic missense mutation, producing the variant p.Lys1665Asn, of unknown pathogenicity. We have modeled this novel missense mutation, as well as a previously described missense mutation p.Met1159Ile, and assessed their effect on ATR function. Interestingly, our data indicate that both missense mutations have no direct effect on protein function, but rather result in defective ATR splicing. These results emphasize the importance of splicing mutations in Seckel Syndrome.

ATR activates the S-M checkpoint during unperturbed growth to ensure sufficient replication prior to mitotic onset.

Cells must accurately replicate and segregate their DNA once per cell cycle in order to successfully transmit genetic information. During S phase in the presence of agents that cause replication stress, ATR-dependent checkpoints regulate origin firing and the replication machinery as well as prevent untimely mitosis. Here, we investigate the role of ATR during unperturbed growth in vertebrate cells. In the absence of ATR, individual replication forks progress more slowly, and an increased number of replication origins are activated. These cells also enter mitosis early and divide more rapidly, culminating in chromosome bridges and laggards at anaphase, failed cytokinesis, and cell death. Interestingly, cell death can be rescued by prolonging mitosis with partial inhibition of the mitotic cyclin-dependent kinase 1. Our data indicate that one of the essential roles of ATR during normal growth is to minimize the level of unreplicated DNA before the onset of mitosis.