Biology subjects

Cejka, P.

Publications and source records attributed to Cejka, P..

23 records · Page 2Linked to original sources

bioRxiv2019

Identification of MCM8IP, an interactor of MCM8-9 and RPA1 that promotes homologous recombination and DNA synthesis in response to DNA damage

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. HR is carried out by a complex network of DNA repair factors. Here we identify C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a novel DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we identify a direct interaction with MCM8-9, a putative helicase complex mutated in Primary Ovarian Insufficiency, that is crucial for MCM8IPs ability to promote resistance to DNA damaging agents. In addition to its association with MCM8-9, MCM8IP also binds directly to RPA1. We show that the interactions of MCM8IP with both MCM8-9 and RPA are required to maintain replication fork progression in response to treatment with crosslinking agents. Collectively, our work identifies MCM8IP as a key regulator of DNA damage-associated DNA synthesis during DNA recombination and replication.

genetics↗

bioRxiv2019

Removal of Spo11 from meiotic DNA breaks in vitro but not in vivo by Tyrosyl DNA Phosphodiesterase 2

Meiotic recombination events are initiated by DNA double-strand breaks (DSBs) created by the topoisomerase-like protein, Spo11. Similar to type-II topoisomerases, Spo11 becomes covalently linked to the 5' ends generated on each side of the DSB. Whilst Spo11-oligos--the product of nucleolytic removal by Mre11--have been detected in a number of biological systems, the lifetime of the covalent Spo11-DSB precursor has not been systematically determined and may be subject to alternative processing reactions. Here we explore the activity of human Tyrosyl DNA Phosphodiesterase, TDP2, on Spo11-DSBs isolated from S. cerevisiae cells. We demonstrate that TDP2 can remove Spo11 from natural ssDNA-oligos, and dsDNA ends even when in the presence of excess competitor genomic DNA. Interestingly, TDP2-processed Spo11-DSBs are refractory to resection by Exo1, suggesting that ssDNA generated by Mre11 may be essential in vivo to facilitate resection-dependent HR at Spo11-DSBs even if TDP2 were active. Moreover, although TDP2 can remove Spo11 peptides in vitro, TDP2 was unable to remove Spo11 in vivo--unlike during the repair of topoisomerase-induced DNA lesions. These results suggest that Spo11-DNA, but not topoisomerase-DNA cleavage complexes, are inaccessible to the TDP2 enzyme, perhaps due to occlusion by higher order protein complexes resident at sites of meiotic recombination.

molecular biology↗

bioRxiv2019

Competing interactions modulate the activity of Sgs1 during DNA end resection

DNA double-strand break repair by homologous recombination employs long-range resection of the 5 DNA ends at the break points. In Saccharomyces cerevisiae, this process can be performed by the RecQ helicase Sgs1 and the helicase-nuclease Dna2. Though functional interplay has been shown, it remains unclear whether and how the proteins cooperate on the molecular level. Here, we resolved the dynamics of DNA unwinding by Sgs1 at the single molecule level and investigated its regulation by Dna2, the single-stranded DNA binding protein RPA and the Top3-Rmi1 complex. We found that Dna2 modulates the velocity of Sgs1, indicating that during end resection the proteins form a physical complex and couple their activities. Sgs1 unwinds DNA and feeds single-stranded DNA to Dna2 for degradation. RPA is found to regulate the processivity and the affinity of Sgs1 to the DNA fork, while Top3-Rmi1 modulated the velocity of Sgs1. We think that the differential regulation of the Sgs1 activity by its protein partners is important to allow diverse cellular functions of Sgs1 during the maintenance of genome stability.

biochemistry↗

bioRxiv2018

The crossover function of MutSγ is activated via Cdc7-dependent stabilization of Msh4

The MutS{gamma} complex, Msh4-Msh5, binds DNA joint-molecule (JM) intermediates during homologous recombination to promote crossing over and accurate chromosome segregation at the first division of meiosis. MutS{gamma} facilitates the formation and biased resolution of crossover-specific JM intermediates called double Holliday junctions. Here we show that these activities are governed by regulated proteasomal degradation. MutS{gamma} is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable. Activation of MutS{gamma} requires the Dbf4-dependent kinase, Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Phosphorylated Msh4 is chromatin bound and requires DNA strand exchange and chromosome synapsis, implying that DDK specifically targets MutS{gamma} that has already bound nascent JMs. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossover control.

genetics↗

bioRxiv2018

A role for RNA and DNA:RNA hybrids in the modulation of DNA repair by homologous recombination

DNA double-strand breaks (DSBs) are toxic DNA lesions which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1 and promote its recruitment to DSBs. We also show that RNase H2 is in a complex with the HR proteins BRCA1, PALB2, BRCA2, and RAD51, and that it localizes to DSBs in the S/G2 cell-cycle phase. BRCA2 controls DNA:RNA hybrid levels at DSBs by mediating RNase H2 recruitment and, therefore, hybrids degradation. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.

cell biology↗