Nicolas Paquet

Nucleophosmin: from structure and function to disease development

Nucleophosmin (NPM1) is a critical cellular protein that has been implicated in a number of pathways including mRNA transport, chromatin remodeling, apoptosis and genome stability. NPM1 function is a critical requirement for normal cellular biology as is underlined in cancer where NPM1 is commonly overexpressed, mutated, rearranged and sporadically deleted. Consistent with a multifunctional role within the cell, NPM1 can function not only as a proto-oncogene but also as a tumor suppressor. The aim of this review is to look at the less well-described role of NPM1 in the DNA repair pathways as well as the role of NPM1 in the regulation of apoptosis and its mutation in cancers.

A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 helicase to interact with RAD51 recombinase for genome stabilization

Background: The BRC repeat is essential for BRCA2 to bind RAD51 and promote homologous recombination. Results: A BRC repeat variant is essential for RECQL5 to bind RAD51 and suppress homologous recombination. Conclusion: The BRC repeat can be utilized to either promote or suppress homologous recombination. Significance: Discovery of multiple functions of the BRC repeat is important for understanding regulation of homologous recombination. The BRC repeat is a structural motif in the tumor suppressor BRCA2 (breast cancer type 2 susceptibility protein), which promotes homologous recombination (HR) by regulating RAD51 recombinase activity. To date, the BRC repeat has not been observed in other proteins, so that its role in HR is inferred only in the context of BRCA2. Here, we identified a BRC repeat variant, named BRCv, in the RECQL5 helicase, which possesses anti-recombinase activity in vitro and suppresses HR and promotes cellular resistance to camptothecin-induced replication stress in vivo. RECQL5-BRCv interacted with RAD51 through two conserved motifs similar to those in the BRCA2-BRC repeat. Mutations of either motif compromised functions of RECQL5, including association with RAD51, inhibition of RAD51-mediated D-loop formation, suppression of sister chromatid exchange, and resistance to camptothecin-induced replication stress. Potential BRCvs were also found in other HR regulatory proteins, including Srs2 and Sgs1, which possess anti-recombinase activities similar to that of RECQL5. A point mutation in the predicted Srs2-BRCv disrupted the ability of the protein to bind RAD51 and to inhibit D-loop formation. Thus, BRC is a common RAD51 interaction module that can be utilized by different proteins to either promote HR, as in the case of BRCA2, or to suppress HR, as in RECQL5.

Organization and Expression of the GSK3/Shaggy Kinase Gene Family in the Moss Physcomitrella patens Suggest Early Gene Multiplication in Land Plants and an Ancestral Response to Osmotic Stress

GSK3/Shaggy kinases are involved in a wide range of fundamental processes in animal development and metabolism. In angiosperm plants, these kinases are encoded by moderate-sized gene families, which appear to have a complex set of functions. Here, we present the characterization of five members of the GSK3/Shaggy gene family in the bryophyte Physcomitrella patens. The P. patens GSK3/Shaggy kinases (PpSK) are organized in a group of closely related paralogues with respect to their gene sequence and structure. Indeed, a phylogenetic analysis of the GSK3/Shaggy kinase sequences from plants and animals showed that the five PpSK proteins are monophyletic, and closer to subgroups I and IV described in angiosperms. Expression analyses performed by quantitative real-time RT-PCR on a wide range of growing conditions showed that PpSK genes responded only to either desiccation, PEG or sorbitol. As demonstrated by both inductions of marker genes and protonemal cell plasmolyses, these treatments resulted in a hyperosmotic stress. Altogether, these data suggest that (1) GSK3/Shaggy kinase gene multiplication occurred early in plant evolution, before the separation between bryophytes and vascular plants, and (2) both gene loss and duplication occurred in the ancestor of P. patens along with functional gene diversification in angiosperms. However, conservation of the transcriptional responses between Physcomitrella and Arabidopsis suggests the identification of an ancestral response of the GSK3/Shaggy kinases genes to osmotic stress.

A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation.

Thymine DNA glycosylase (TDG) functions in base excision repair, a DNA repair pathway that acts in a lesion-specific manner to correct individual damaged or altered bases. TDG preferentially catalyzes the removal of thymine and uracil paired with guanine, and is also active on 5-fluorouracil (5-FU) paired with adenine or guanine. The rs4135113 single nucleotide polymorphism (SNP) of TDG is found in 10% of the global population. This coding SNP results in the alteration of Gly199 to Ser. Gly199 is part of a loop responsible for stabilizing the flipped abasic nucleotide in the active site pocket. Biochemical analyses indicate that G199S exhibits tighter binding to both its substrate and abasic product. The persistent accumulation of abasic sites in cells expressing G199S leads to the induction of double-strand breaks (DSBs). Cells expressing the G199S variant also activate a DNA damage response. When expressed in cells, G199S induces genomic instability and cellular transformation. Together, these results suggest that individuals harboring the G199S variant may have increased risk for developing cancer.

The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16–18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

hSSB1 (NABP2/OBFC2B) is regulated by oxidative stress

The maintenance of genome stability is an essential cellular process to prevent the development of diseases including cancer. hSSB1 (NABP2/ OBFC2A) is a critical component of the DNA damage response where it participates in the repair of double-strand DNA breaks and in base excision repair of oxidized guanine residues (8-oxoguanine) by aiding the localization of the human 8-oxoguanine glycosylase (hOGG1) to damaged DNA. Here we demonstrate that following oxidative stress, hSSB1 is stabilized as an oligomer which is required for hSSB1 to function in the removal of 8-oxoguanine. Monomeric hSSB1 shows a decreased affinity for oxidized DNA resulting in a cellular 8-oxoguanine-repair defect and in the absence of ATM signaling initiation. While hSSB1 oligomerization is important for the removal of 8-oxoguanine from the genome, it is not required for the repair of double-strand DNA-breaks by homologous recombination. These findings demonstrate a novel hSSB1 regulatory mechanism for the repair of damaged DNA.

A structural analysis of DNA binding by hSSB1 (NABP2/OBFC2B) in solution

Single-stranded DNA binding proteins (SSBs) play an important role in DNA processing events such as replication, recombination and repair. Human single-stranded DNA binding protein 1 (hSSB1/NABP2/OBFC2B) contains a single oligosaccharide/oligonucleotide binding (OB) domain followed by a charged C-terminus and is structurally homologous to the SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus. Recent work has revealed that hSSB1 is critical to homologous recombination and numerous other important biological processes such as the regulation of telomeres, the maintenance of DNA replication forks and oxidative damage repair. Since the ability of hSSB1 to directly interact with single-stranded DNA (ssDNA) is paramount for all of these processes, understanding the molecular details of ssDNA recognition is essential. In this study, we have used solution-state nuclear magnetic resonance in combination with biophysical and functional experiments to structurally analyse ssDNA binding by hSSB1. We reveal that ssDNA recognition in solution is modulated by base-stacking of four key aromatic residues within the OB domain. This DNA binding mode differs significantly from the recently determined crystal structure of the SOSS1 complex containing hSSB1 and ssDNA. Our findings elucidate the detailed molecular mechanism in solution of ssDNA binding by hSSB1, a major player in the maintenance of genomic stability.

Structure and vascular tissue expression of duplicated TERMINAL EAR1-like paralogues in poplar

TERMINAL EAR1-like (TEL) genes encode putative RNA-binding proteins only found in land plants. Previous studies suggested that they may regulate tissue and organ initiation in Poaceae. Two TEL genes were identified in both Populus trichocarpa and the hybrid aspen Populus tremula × P. alba, named, respectively, PoptrTEL1-2 and PtaTEL1-2. The analysis of the organisation around the PoptrTEL genes in the P. trichocarpa genome and the estimation of the synonymous substitution rate for PtaTEL1-2 genes indicate that the paralogous link between these two Populus TEL genes probably results from the Salicoid large-scale gene-duplication event. Phylogenetic analyses confirmed their orthology link with the other TEL genes. The expression pattern of both PtaTEL genes appeared to be restricted to the mother cells of the plant body: leaf founder cells, leaf primordia, axillary buds and root differentiating tissues, as well as to mother cells of vascular tissues. Most interestingly, PtaTEL1-2 transcripts were found in differentiating cells of secondary xylem and phloem, but probably not in the cambium itself. Taken together, these results indicate specific expression of the TEL genes in differentiating cells controlling tissue and organ development in Populus (and other Angiosperm species).

17P Examination of EXOSC4 as a new prognostic marker and a novel therapeutic avenue in lung adenocarcinoma

Although prevention and early detection of the disease greatly improved over the past few years, lung cancer remains the leading cause of cancer deaths. In order to be able to treat a larger population, we are in urgent need for novel treatments. While it is known that DNA repair genes play a major role in the oncogenic transformation, they also represent a weakness of cancers that constitute a therapeutic opportunity. To identify novel DNA repair genes implicated in Lung cancers, we conducted an in silico investigation to identify genes co-regulated with two DNA repair factors, BRCA2 and hSSB1. This approach allowed for the identification of EXOSC4, a component of the RNA Exosome machinery, as a potential factor involved in the maintenance of genome stability and that is deregulated in lung cancer.

Novel insight into the composition of human single-stranded DNA-binding protein 1 (hSSB1)-containing protein complexes

BackgroundSingle-stranded DNA-binding proteins are essential cellular components required for the protection, metabolism and processing of single-stranded DNA. Human single-stranded DNA-binding protein 1 (hSSB1) is one such protein, with described roles in genome stability maintenance and transcriptional regulation. As yet, however, the mechanisms through which hSSB1 functions and the binding partners with which it interacts remain poorly understood.ResultsIn this work, hSSB1 was immunoprecipitated from cell lysate samples that had been enriched for non-soluble nuclear proteins and those associating with hSSB1 identified by mass spectrometry. In doing so, 334 potential hSSB1-associating proteins were identified, with known roles in a range of distinct biological processes. Unexpectedly, whilst hSSB1 has largely been studied in a genome stability context, few other DNA repair or replication proteins were detected. By contrast, a large number of proteins were identified with roles in mRNA metabolism, reflecting a currently emerging area of hSSB1 study. In addition, numerous proteins were detected that comprise various chromatin-remodelling complexes.ConclusionsThese findings provide new insight into the binding partners of hSSB1 and will likely function as a platform for future research.