Day, D. | Search bioRxiv

bioRxiv2018

Histone deposition plays an active role in recombination-dependent replication to balance genome2 stability upon replication stress

Replication stress poses a serious threat to genome and epigenome stability. Recombination-Dependent-Replication (RDR) ensures DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways during DNA repair processes, crosstalk coupling RDR and chromatin assembly is largely unexplored. Here, we addressed the contribution of chromatin assembly to replication stress in fission yeast. We expressed a mutated histone (H3-H113D) to genetically impair replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition is required for the completion of RDR. Histone deposition prevents joint-molecules from Rqh1-dependent disassembly, a crosstalk contributing to cell survival upon replication stress but channeling RDR towards deleterious events. Asf1 and CAF-1 act in RDR and CAF-1 recruitment to DNA synthesis associated to RDR requires the HR factor Rad52. Our data establish that CAF-1 counteracts Rqh1 activity at sites of replication stress by promoting repair synthesis-coupled histone deposition. These results demonstrate that histone deposition plays an active role in fine-tuning RDR, a benefit counterbalanced by stabilizing at-risk joint molecules for genome stability.

genetics↗

bioRxiv2018

Optimizing CRISPR/Cas9 System to Precisely Model Plasminogen Activator Inhibitor-1 Point Mutations in Mice

CRISPR/Cas9 has become a powerful genome editing tool in recent years. CRISPR/Cas9 can be utilized to not only efficiently generate knock out models in various organisms, but also to precisely model human disease or variants to study gene function and develop therapies. However, the latter remains challenging because of low knock-in (KI) efficiency. In this study, precise gene editing modeling plasminogen activator inhibitor-1 (PAI-1) -tissue plasminogen activator (tPA) binding deficiency and PAI-1-vitronectin binding deficiency were generated respectively in mice. Optimization of single guide RNAs (sgRNA) and repair templates, and utilization of restriction fragment length polymorphism (RFLP) to detect KI events are described. Injection of sgRNA/Cas9/single-stranded oligodeoxynucleotide (ssODN) into mouse zygotes resulted in homozygous changes of two silent mutations and changed Arg369>Ala, which abolishes PAI-1 inhibitory activity against tPA. Targeting Arg124 and Gln146 simultaneously involved in vitronectin binding proved to be challenging. However, we successfully generated these relatively distant mutations (23 amino acids apart) seamlessly. Generation of the Arg124 mutation alone was achieved with over 60% efficiency along with the integration of a restriction site, compared to the relatively low double mutation frequency. In summary, our data indicates that the distance between desired mutations and CRISPR-induced double-stranded break (DSB) site is the most critical factor for achieving high efficiency in precise gene modification.

genetics↗