Cooperative motion of a key positively charged residue and metal ions for DNA replication catalyzed by human DNA Polymerase-eta

Trans-lesion synthesis polymerases, like DNA Polymerase-eta (Pol-eta), are essential for cell survival. Pol-eta bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-eta function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-eta catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-eta for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases.

How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle

Florence Magnin

Adeno-associated virus (AAV) is a small DNA virus belonging to the family of the parvoviridae. AAV infects the human population, however no disease has been associated with this virus. On the contrary, AAV was reported to have oncosuppressive activities. The AAV genome contains two genes: the Rep gene encoding four non-structural proteins (Rep78, 68, 52 and 40) and the Cap gene encoding the three capsid proteins. Rep78 is implicated in transcription, replication and site-specific integration of the viral DNA. It has been shown to block the cell cycle in G1, G2 and S phase with a complete inhibition of DNA synthesis. To be able to induce such a strong cell cycle arrest, Rep78 affects several proteins implicated in the regulation of the cell cycle, such as the family of the cell division cycle 25 (Cdc25) phosphatases. Rep78 has been shown to inactivate Cdc25A by binding to it and preventing it from accessing its substrates. Cdc25B shares conserved domains with Cdc25A and moreover has an important role in cell cycle regulation, especially in giving the signal for entry into mitosis. Thus, in this work, the interaction between Cdc25B and Rep78 was examined in more detail. Rep78 was found to bind to Cdc25B and to alter its intracellular localisation. Cdc25B is nuclear, but has to migrate into the cytoplasm at the end of the G2 phase to activate proteins needed for the entry into mitosis. The expression of Rep78, which is nuclear, leads to the sequestration of Cdc25B in the nucleus. Moreover, Rep78 by interacting with Cdc25B not only changes its localisation, but also inhibits its phosphatase activity. Our model is that Rep78 interacts with Cdc25B and thus retains it in the nucleus. This sequestration of Cdc25B in the nucleus prevents it from activating the proteins in the cytoplasm needed to give the entry signal into mitosis, thus contributing to the G2/M arrest induced by Rep78. Moreover, the inhibition of Cdc25B phosphatase activity by Rep78 may also contribute to the cell cycle block. Rep78 also interacts with a number of cellular proteins implicated in DNA replication, DNA repair or recombination. Interactions between Rep78 and mini-chromosome maintenance 3 (MCM3), DNA polymerases α and δ, proliferating cell nuclear antigen (PCNA), Rad50 and Rad51 were identified in this work as well as possible interactions between Rep78 and Ataxia telangiectasia mutated (ATM) or Ataxia telangiectasia and Rad3 related (ATR) proteins. These interactions may contribute to AAV genome replication as well as its integration into the host DNA.

EPFL2009

Understanding the Effect of Magnesium Ion Concentration on the Catalytic Activity of Ribonuclease H through Computation: Does a Third Metal Binding Site Modulate Endonuclease Catalysis?

Matteo Dal Peraro

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg2+ concentration. Moreover, in RNase H, the glutamate residue E188 has been shown to be essential for full enzymatic activation, regardless of the Mg2+ concentration. The catalytic center is known to contain two Mg2+ ions, and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg2+. Importantly, the presence of a third Mg2+ ion, bound to the second-shell ligand E188, is a persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations, depending on the concentration of Mg2+. At standard concentration, a third Mg2+ is found in the catalytic pocket, but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg2+ ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg2+ ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A and also supports the key role of E188 in localizing the third Mg2+ ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calculations support the hypothesis that RNase H can accommodate three divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.

2010

Computational Studies of the Proton-Coupled Metal Ion Transport in the SLC11/NRAMP Family of Transporters

Maria Letizia Merlini

In the last years, it has been demonstrated a link between the overload of metal ions inside nervous system cells and the onset of severe neurodegenerative diseases. This prompted the investigation of the structural and functional properties of transporters responsible for the uptake and regulation of metal ions inside cells. In particular, members of the Nramp/SLC11 ("natural resistance-associated macrophage proteins"/"solute carrier 11") family of transporters tightly regulate the influx of divalent transition-metal ions across cellular membranes contributing to cellular homeostasis. In these secondary active transporters, the translocation of divalent transition metal ions from the extracellular matrix to the cellular cytoplasm is coupled to proton transport (symport). The co-transport is realized using an alternate access mechanism in which the protein is alternately open towards the extracellular matrix, ("outward facing conformation", OFC) or towards the cellular cytoplasm ("inward facing conformation", IFC).Although a wide range of functional and structural studies provided fundamental insights on the proton-coupled transport, most mechanistic details are still unknown. As an example, the coordination sphere of the metal ion in the inward-facing conformation of the protein has only been partially resolved by crystallographic experiments and later attempts to provide comprehensive descriptions led to contradictory models. Furthermore, several experimental evidence proved that proton uniport in the absence of the metal ion, is also possible, suggesting an unusual symporter behaviour for Nramps, in which the two substrates are not tightly coupled. Despite the recent advances in the comprehension of Nramps proton transport, the underlying mechanistic details remain still ambiguous, as demonstrated by inconsistent mechanistic models proposed during the last years.In this thesis, computational methods have been used in order to provide new molecular level insights in the proton-coupled divalent transition metal ions transport performed by Nramps. In particular, a combination of classical molecular dynamics (MD) and QM/MM ab initio MD methods have been applied in order to refine the partially resolved coordination sphere of Mn2+ in the active site of Staphylococcus Capitis Divalent Metal-ion Transporter (ScaDMT), representing the only available crystal structure of a substrate-bound member of the family in an IFC. This QM/MM multiscale approach, using an accurate quantum treatment of the active site and a classical treatment of the surroundings, allowed for state-of-the-art calculations of the entire system that led to the unequivocal identification of the metal's coordination sphere. Furthermore, MD simulations have been performed for both the IFC ScaDMT and Eremococcus Coleocola DMT (EcoDMT), whose crystal structure has been obtained in OFC. Since experimental evidences proved that proton transport can occur even in the absence of the metal ion substrate, we performed simulations of both proteins in their apo forms. These simulations allowed us to characterize four amino acidic residues identified as likely key players in the proton transport process and to determine which role they assume in the process. Finally, we were able to identify the likely primary proton acceptor and proposed a potential route followed by protons in their influx toward the intracellular cytoplasm.

EPFL2022