Hélène Launay

Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination

Inherited mutations in human PALB2 are associated with a predisposition to breast and pancreatic cancers. PALB2′s tumor-suppressing effect is thought to be based on its ability to facilitate BRCA2′s function in homologous recombination. However, the biochemical properties of PALB2 are unknown. Here we show that human PALB2 binds DNA, preferentially D-loop structures, and directly interacts with the RAD51 recombinase to stimulate strand invasion, a vital step of homologous recombination. This stimulation occurs through reinforcing biochemical mechanisms, as PALB2 alleviates inhibition by RPA and stabilizes the RAD51 filament. Moreover, PALB2 can function synergistically with a BRCA2 chimera (termed piccolo, or piBRCA2) to further promote strand invasion. Finally, we show that PALB2-deficient cells are sensitive to PARP inhibitors. Our studies provide the first biochemical insights into PALB2′s function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair.

Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy

Protein folding in living cells is inherently coupled to protein synthesis and chain elongation. There is considerable evidence that some nascent chains fold into their native structures in a cotranslational manner before release from the ribosome, but, despite its importance, a detailed description of such a process at the atomic level remains elusive. We show here at a residue-specific level that a nascent protein chain can reach its native tertiary structure on the ribosome. By generating translation-arrested ribosomes in which the newly synthesized polypeptide chain is selectively 13C/15N-labeled, we observe, using ultrafast NMR techniques, a large number of resonances of a ribosome-bound nascent chain complex corresponding to a pair of C-terminally truncated immunoglobulin (Ig) domains. Analysis of these spectra reveals that the nascent chain adopts a structure in which a native-like N-terminal Ig domain is tethered to the ribosome by a largely unfolded and highly flexible C-terminal domain. Selective broadening of resonances for a group of residues that are colocalized in the structure demonstrates that there are specific but transient interactions between the ribosome and the N-terminal region of the folded Ig domain. These findings represent a step toward a detailed structural understanding of the cellular processes of cotranslational folding.

A mutation in T7 RNA polymerase that facilitates promoter clearance

Like multisubunit RNA polymerases (RNAPs), T7 RNAP frequently releases its transcript over the initial 8-12 transcribed nucleotides, when it still contacts the promoter. This abortive cycling, which is most prominent with initial sequences that deviate from those of T7 late genes, eventually compromises productive transcription. Starting from an in vivo situation where transcription of a target gene by T7 RNAP is virtually abolished because of extensive abortive cycling, we have selected a mutation in RNAP that restores target gene expression. In vitro, this mutation (P266L) weakens promoter binding but markedly reduces abortive cycling over a variety of initial sequences by stabilizing the transcription complex at nucleotides 5-8. Other substitutions of P266 have similar effects. X-ray data show that during the transition from initial to elongation complex, the N-terminal region undergoes a major structural switch of which P266 constitutes one of the hinges. How the mutation might facilitate this switch is tentatively discussed. On the practical side, the mutation can significantly improve in vitro transcription, particularly from templates carrying unfavorable initial sequences.

Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy.

The means by which a polypeptide chain acquires its unique 3-D structure is a fundamental question in biology. During its synthesis on the ribosome, a nascent chain (NC) emerges vectorially and will begin to fold in a cotranslational fashion. The complex environment of the cell, coupled with the gradual emergence of the ribosome-tethered NC during its synthesis, imposes conformational restraints on its folding landscape that differ from those placed on an isolated protein when stimulated to fold following denaturation in solution. To begin to examine cotranslational folding as it would occur within a cell, we produce highly selective, isotopically labeled NCs bound to isotopically silent ribosomes in vivo. We then apply NMR spectroscopy to study, at a residue specific level, the conformation of NCs consisting of different fractional lengths of the polypeptide chain corresponding to a given protein. This combined approach provides a powerful means of generating a series of snapshots of the folding of the NC as it emerges from the ribosome. Application of this strategy to the NMR analysis of the progressive synthesis of an Ig-like domain reveals the existence of a partially folded ribosome-bound species that is likely to represent an intermediate species populated during the cotranslational folding process.

A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding

Although detailed pictures of ribosome structures are emerging, little is known about the structural and cotranslational folding properties of nascent polypeptide chains at the atomic level. Here we used solution-state NMR spectroscopy to define a structural ensemble of a ribosome–nascent chain complex (RNC) formed during protein biosynthesis in Escherichia coli, in which a pair of immunoglobulin-like domains adopts a folded N-terminal domain (FLN5) and a disordered but compact C-terminal domain (FLN6). To study how FLN5 acquires its native structure cotranslationally, we progressively shortened the RNC constructs. We found that the ribosome modulates the folding process, because the complete sequence of FLN5 emerged well beyond the tunnel before acquiring native structure, whereas FLN5 in isolation folded spontaneously, even when truncated. This finding suggests that regulating structure acquisition during biosynthesis can reduce the probability of misfolding, particularly of homologous domains.

Protein folding on the ribosome studied using NMR spectroscopy

NMR spectroscopy is a powerful tool for the investigation of protein folding and misfolding, providing a characterization of molecular structure, dynamics and exchange processes, across a very wide range of timescales and with near atomic resolution. In recent years NMR methods have also been developed to study protein folding as it might occur within the cell, in a de novo manner, by observing the folding of nascent polypeptides in the process of emerging from the ribosome during synthesis. Despite the 2.3 MDa molecular weight of the bacterial 70S ribosome, many nascent polypeptides, and some ribosomal proteins, have sufficient local flexibility that sharp resonances may be observed in solution-state NMR spectra. In providing information on dynamic regions of the structure, NMR spectroscopy is therefore highly complementary to alternative methods such as X-ray crystallography and cryo-electron microscopy, which have successfully characterized the rigid core of the ribosome particle. However, the low working concentrations and limited sample stability associated with ribosome-nascent chain complexes means that such studies still present significant technical challenges to the NMR spectroscopist. This review will discuss the progress that has been made in this area, surveying all NMR studies that have been published to date, and with a particular focus on strategies for improving experimental sensitivity.

Lactococcal phage p2 ORF35‐Sak3 is an ATPase involved in DNA recombination and AbiK mechanism

Virulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35p2, also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single‐strand binding protein as well as its membership to a superfamily of single‐strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35p2 form a hexameric ring‐like structure that is often found in proteins with a conserved RecA nucleotide‐binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35p2 interacts preferentially with single‐stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35p2 is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA‐mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35p2 is a novel SSAP stimulating homologous recombination.

Structure and function of phage p2 ORF34p2, a new type of single-stranded DNA binding protein

Lactococcus lactis, a Gram‐positive bacterium widely used by the dairy industry, is subject to infection by a diverse population of virulent phages, predominantly by those of the 936 group, including the siphovirus phage p2. Confronted with the negative impact of phage infection on milk fermentation, the study of the biology of lactococcal provides insight from applied and fundamental perspectives. We decided to characterize the product of the orf34 gene from lactococcus phage p2, which was considered as a candidate single‐stranded DNA binding protein (SSB) due to its localization downstream of a gene coding for a single‐strand annealing protein. Two‐dimensional gel electrophoresis showed that ORF34p2 is expressed in large amounts during the early phases of phage infection, suggesting an important role in this process. Gel‐shift assays, surface plasmon resonance and atomic force microscopy demonstrated that ORF34p2 interacts with single‐strand DNA with nanomolar affinity. We also determined the crystal structure of ORF34p2 and showed that it bears a variation of the typical oligonucleotide/oligosaccharide binding‐fold of SSBs. Finally, we found that ORF34p2 is able to stimulate Escherichia coli RecA‐mediated homologous recombination. The specific structural and biochemical properties that distinguish ORF34p2 from other SSB proteins are discussed.

Deciphering the function of lactococcal phage ul36 Sak domains.

Virulent phages are responsible for milk fermentation failures in the dairy industry, due to their ability to infect starter cultures containing strains of Lactococcus lactis. Single-strand annealing proteins (SSAPs) have been found in several lactococcal phages, among which Sak in the phage ul36. Sak has been recently shown to be a functional homolog of the human protein RAD52, involved in homologous recombination. A comparison between full-length Sak and its N- and C-terminal domains was carried out to elucidate functional characteristics of each domain. We performed HPLC-SEC, AFM and SPR experiments to evaluate oligomerization states and compare the affinities to DNA. We have shown that the N-terminal domain (1-171) is essential and sufficient for oligomerization and binding to DNA, while the C-terminal domain (172-252) does not bind DNA nor oligomerize. Modelisation of Sak N-terminal domain suggests that DNA may bind a positively charged crevice that runs external to the ring. Annealing and stimulation of RecA strand exchange indicate that only the N-terminal domain is capable of single-strand annealing and both domains do not stimulate the RecA strand exchange reaction. We propose that Sak N-terminus is involved in DNA binding and annealing while the C-terminus may serve to contact Sak partners.

An HflX-Type GTPase from Sulfolobus solfataricus Binds to the 50S Ribosomal Subunit in All Nucleotide-Bound States

HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint (1)H-(15)N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins.