NBS1 recruits RAD18 via a RAD6-like domain and regulates Pol η-dependent translesion DNA synthesis

Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis.     

DNA methylation of the promoter region at the CREB1 binding site is a mechanism for the epigenetic regulation of brain-specific PKMζ

Protein kinase M zeta, PKMζ, is a brain enriched kinase with a well characterized role in Long-Term Potentiation (LTP), the activity-dependent strengthening of synapses involved in long-term memory formation. However, little is known about the molecular mechanisms that maintain the tissue specificity of this kinase. Here, we characterized the epigenetic factors, mainly DNA methylation, regulating PKMζ expression in the human brain. The PRKCZ gene has an upstream promoter regulating Protein kinase C ζ (PKCζ), and an internal promoter driving PKMζ expression. A demethylated region, including a canonical CREB binding site, situated at the internal promoter was only observed in human CNS tissues. The induction of site-specific hypermethylation of this region resulted in decreased CREB1 binding and downregulation of PKMζ expression. Noteworthy, CREB binding sites were absent in the upstream promoter of PRKCZ locus, suggesting a specific mechanism for regulating PKMζ expression. These observations were validated using a system of human neuronal differentiation from induced pluripotent stem cells (iPSCs). CREB1 binding at the internal promoter was detected only in differentiated neurons, where PKMζ is expressed. The same epigenetic mechanism in the context of CREB binding site was identified in other genes involved in neuronal differentiation and LTP. Additionally, aberrant DNA hypermethylation at the internal promoter was observed in cases of Alzheimer's disease, correlating with decreased expression of PKMζ in patient brains. Altogether, we present a conserved epigenetic mechanism regulating PKMζ expression and other genes enhanced in the CNS with possible implications in neuronal differentiation and Alzheimer's disease.     

The 14-3-3ζ protein binds to the cell adhesion molecule L1, promotes L1 phosphorylation by CKII and influences L1-dependent neurite outgrowth

Background

The cell adhesion molecule L1 is crucial for mammalian nervous system development. L1 acts as a mediator of signaling events through its intracellular domain, which comprises a putative binding site for 14-3-3 proteins. These regulators of diverse cellular processes are abundant in the brain and preferentially expressed by neurons. In this study, we investigated whether L1 interacts with 14-3-3 proteins, how this interaction is mediated, and whether 14-3-3 proteins influence the function of L1.

Methodology/Principal Findings

By immunoprecipitation, we demonstrated that 14-3-3 proteins are associated with L1 in mouse brain. The site of 14-3-3 interaction in the L1 intracellular domain (L1ICD), which was identified by site-directed mutagenesis and direct binding assays, is phosphorylated by casein kinase II (CKII), and CKII phosphorylation of the L1ICD enhances binding of the 14-3-3 zeta isoform (14-3-3ζ). Interestingly, in an in vitro phosphorylation assay, 14-3-3ζ promoted CKII-dependent phosphorylation of the L1ICD. Given that L1 phosphorylation by CKII has been implicated in L1-triggered axonal elongation, we investigated the influence of 14-3-3ζ on L1-dependent neurite outgrowth. We found that expression of a mutated form of 14-3-3ζ, which impairs interactions of 14-3-3ζ with its binding partners, stimulated neurite elongation from cultured rat hippocampal neurons, supporting a functional connection between L1 and 14-3-3ζ.

Conclusions/Significance

Our results suggest that 14-3-3ζ, a novel direct binding partner of the L1ICD, promotes L1 phosphorylation by CKII in the central nervous system, and regulates neurite outgrowth, an important biological process triggered by L1.     

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase α and is required for cell cycle progression in G2?/?M

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cΔ) shows slow growth at low temperature (15° C), while at 30° C and 37° C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Poll, the catalytic subunit of DNA polymerase α. The pol1-1 /yjr043cΔ double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cΔ. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cΔ mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication. Received: 22 July 1998 / Accepted: 22 September 1998