SMK-1/PPH-4.1–mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

During early embryogenesis in Caenorhabditis elegans, the ATL-1–CHK-1 (ataxia telangiectasia mutated and Rad3 related–Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1–PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context

The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication

The initiation of eukaryotic DNA replication involves origin recruitment and activation of the MCM2-7 complex, the putative replicative helicase. Mini-chromosome maintenance (MCM)2-7 recruitment to origins in G1 requires origin recognition complex (ORC), Cdt1, and Cdc6, and activation at G1/S requires MCM10 and the protein kinases Cdc7 and S-Cdk, which together recruit Cdc45, a putative MCM2-7 cofactor required for origin unwinding. Here, we show that the Xenopus BRCA1 COOH terminus repeat–containing Xmus101 protein is required for loading of Cdc45 onto the origin. Xmus101 chromatin association is dependent on ORC, and independent of S-Cdk and MCM2-7. These results define a new factor that is required for Cdc45 loading. Additionally, these findings indicate that the initiation complex assembly pathway bifurcates early, after ORC association with the origin, and that two parallel pathways, one controlled by MCM2-7, and the other by Xmus101, cooperate to load Cdc45 onto the origin

Systematic, RNA-Interference-Mediated Identification of mus-101 Modifier Genes in Caenorhabditis elegans

The Mus101 family of chromosomal proteins, identified initially in Drosophila, is widely conserved and has been shown to function in a variety of DNA metabolic processes. Such functions include DNA replication, DNA damage repair, postreplication repair, damage checkpoint activation, chromosome stability, and chromosome condensation. Despite its conservation and widespread involvement in chromosome biogenesis, very little is known about how Mus101 is regulated and what other proteins are required for Mus101 to exert its functions. To learn more about Mus101, we have initiated an analysis of the protein in C. elegans. Here, we show that C. elegans mus-101 is an essential gene, that it is required for DNA replication, and that it also plays an important role in the DNA damage response. Furthermore, we use RNA interference (RNAi)-mediated reverse genetics to screen for genes that modify a mus-101 partial loss-of-function RNAi phenotype. Using a systematic approach toward modifier gene discovery, we have found five chromosome I genes that modify the mus-101 RNAi phenotype, and we go on to show that one of them encodes an E3 SUMO ligase that promotes SUMO modification of MUS-101 in vitro. These results expand our understanding of MUS-101 regulation and show that genetic interactions can be uncovered using screening strategies that rely solely on RNAi

Spatiotemporal expression profiling of proteins in rat sciatic nerve regeneration using reverse phase protein arrays

Abstract Background Protein expression profiles throughout 28 days of peripheral nerve regeneration were characterized using an established rat sciatic nerve transection injury model. Reverse phase protein microarrays were used to identify the spatial and temporal expression profile of multiple proteins implicated in peripheral nerve regeneration including growth factors, extracellular matrix proteins, and proteins involved in adhesion and migration. This high-throughput approach enabled the simultaneous analysis of 3,360 samples on a nitrocellulose-coated slide. Results The extracellular matrix proteins collagen I and III, laminin gamma-1, fibronectin, nidogen and versican displayed an early increase in protein levels in the guide and proximal sections of the regenerating nerve with levels at or above the baseline expression of intact nerve by the end of the 28 day experimental course. The 28 day protein levels were also at or above baseline in the distal segment however an early increase was only noted for laminin, nidogen, and fibronectin. While the level of epidermal growth factor, ciliary neurotrophic factor and fibroblast growth factor-1 and -2 increased throughout the experimental course in the proximal and distal segments, nerve growth factor only increased in the distal segment and fibroblast growth factor-1 and -2 and nerve growth factor were the only proteins in that group to show an early increase in the guide contents. As expected, several proteins involved in cell adhesion and motility; namely focal adhesion kinase, N-cadherin and β-catenin increased earlier in the proximal and distal segments than in the guide contents reflecting the relatively acellular matrix of the early regenerate. Conclusions In this study we identified changes in expression of multiple proteins over time linked to regeneration of the rat sciatic nerve both demonstrating the utility of reverse phase protein arrays in nerve regeneration research and revealing a detailed, composite spatiotemporal expression profile of peripheral nerve regeneration.</p