Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5′ overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.     

PARP–nucleic acid interactions: Allosteric signaling,PARP inhibitor types,DNA bridges,and viral RNA surveillance

PARP enzymes create ADP-ribose modifications to regulate multiple facets of human biology, and some prominent PARP family members are best known for the nucleic acid interactions that regulate their activities and functions. Recent structural studies have highlighted PARP interactions with nucleic acids, in particular for PARP enzymes that detect and respond to DNA strand break damage. These studies build on our understanding of how DNA break detection is linked to the catalysis of ADP-ribose modifications, provide insights into distinct modes of DNA interaction, and shed light on the mechanisms of PARP inhibitor action. PARP enzymes have several connections to RNA biology, including the detection of the genomes of RNA viruses, and recent structural work has highlighted how PARP13/ZAP specifically targets viral genomes enriched in CG dinucleotides.     

PARP Activity Fine-tunes the DNA Replication Choreography of Chk1-depleted Cells

Checkpoint Kinase 1 (Chk1) prevents DNA damage by adjusting the replication choreography in the face of replication stress. Chk1 depletion provokes slow and asymmetrical fork movement, yet the signals governing such changes remain unclear. We sought to investigate whether poly(ADP-ribose) polymerases (PARPs), key players of the DNA damage response, intervene in the DNA replication of Chk1-depleted cells. We demonstrate that PARP inhibition selectively alleviates the reduced fork elongation rates, without relieving fork asymmetry in Chk1-depleted cells. While the contribution of PARPs to fork elongation is not unprecedented, we found that their role in Chk1-depleted cells extends beyond fork movement. PARP-dependent fork deceleration induced mild dormant origin firing upon Chk1 depletion, augmenting the global rates of DNA synthesis. Thus, we have identified PARPs as novel regulators of replication fork dynamics in Chk1-depleted cells.     

Structure Basis for Shaping the Nse4 Protein by the Nse1 and Nse3 Dimer within the Smc5/6 Complex

The Smc5/6 complex facilitates chromosome replication and DNA break repair. Within this complex, a subcomplex composed of Nse1, Nse3 and Nse4 is thought to play multiple roles through DNA binding and regulating ATP-dependent activities of the complex. However, how the Nse1-Nse3-Nse4 subcomplex carries out these multiple functions remain unclear. To address this question, we determine the crystal structure of the Xenopus laevis Nse1-Nse3-Nse4 subcomplex at 1.7 Å resolution and examine how it interacts with DNA. Our structural analyses show that the Nse1-Nse3 dimer adopts a closed conformation and forms three interfaces with a segment of Nse4, forcing it into a Z-shaped conformation. The Nse1-Nse3-Nse4 structure provides an explanation for how the lung disease immunodeficiency and chromosome breakage syndrome-causing mutations could dislodge Nse4 from Nse1-Nse3. Our DNA binding and mutational analyses reveal that the N-terminal and the middle region of Nse4 contribute to DNA interaction and cell viability. Integrating our data with previous crosslink mass spectrometry data, we propose potential roles of the Nse1-Nse3-Nse4 complex in binding DNA within the Smc5/6 complex.     

Sialic acid diversity in the human gut: Molecular impacts and tools for future discovery

Sialic acids are a family of structurally related sugars that are prevalent in mucosal surfaces, including the human intestine. In the gut, sialic acids have diverse biological roles at the interface of the host epithelium and the microbiota. N-acetylneuraminic acid (Neu5Ac), the best studied sialic acid, is a nutrient source for bacteria and, when displayed on the cell surface, a binding site for host immune factors, viruses, and bacterial toxins. Neu5Ac is extensively modified by host and microbial enzymes, and the impacts of Neu5Ac derivatives on host–microbe interactions, and generally on human and microbial biology, remain underexplored. In this mini-review, we highlight recent reports describing how host and microbial proteins differentiate Neu5Ac and its derivatives, draw attention to gaps in knowledge related to sialic acid biology, and suggest cutting-edge methodologies that may expand our appreciation and understanding of Neu5Ac in health and disease.     

Molecular evolution and functional divergence of UDP-hexose 4-epimerases

UDP-glucose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) and/or the interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc) in sugar metabolism. GalEs belong to the short-chain dehydrogenase/reductase superfamily, use a conserved ‘transient keto intermediate’ mechanism and have variable substrate specificity. GalEs have been classified into three groups based on substrate specificity: group 1 prefers UDP-Glc/Gal, group 3 prefers UDP-GlcNAc/GalNAc, and group 2 has comparable activities for both types of the substrates. The phylogenetic relationship and structural basis for the specificities of GalEs revealed possible molecular evolution of UDP-hexose 4-epimerases in various organisms. Based on the recent advances in studies on GalEs and related enzymes, an updated view of their evolutional diversification is presented.     

The Structure of Saccharomyces cerevisiae Arginyltransferase 1 (ATE1)

Eukaryotic post-translational arginylation, mediated by the family of enzymes known as the arginyltransferases (ATE1s), is an important post-translational modification that can alter protein function and even dictate cellular protein half-life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cell division, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. To that end, we have used X-ray crystallography to solve the crystal structure of ATE1 from the model organism Saccharomyces cerevisiae ATE1 (ScATE1) in the apo form. The three-dimensional structure of ScATE1 reveals a bilobed protein containing a GCN5-related N-acetyltransferase (GNAT) fold, and this crystalline behavior is faithfully recapitulated in solution based on size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS) analyses and cryo-EM 2D class averaging. Structural superpositions and electrostatic analyses point to this domain and its domain-domain interface as the location of catalytic activity and tRNA binding, and these comparisons strongly suggest a mechanism for post-translational arginylation. Additionally, our structure reveals that the N-terminal domain, which we have previously shown to bind a regulatory [Fe-S] cluster, is dynamic and disordered in the absence of metal bound in this location, hinting at the regulatory influence of this region. When taken together, these insights bring us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation.     

Interactome Analysis of Human Phospholipase D and Phosphatidic Acid-Associated Protein Network

Mammalian phospholipase D (PLD) enzyme family consists of six members. Among them, PLD1/2/6 catalyzes phosphatidic acid (PA) production, while PLD3/4/5 has no catalytic activities. Deregulation of the PLD-PA lipid signaling has been associated with various human diseases including cancer. However, a comprehensive analysis of the regulators and effectors for this crucial lipid metabolic pathway has not been fully achieved. Using a proteomic approach, we defined the protein interaction network for the human PLD family of enzymes and PA and revealed diverse cellular signaling events involving them. Through it, we identified PJA2 as a novel E3 ubiquitin ligase for PLD1 involved in control of the PLD1-mediated mammalian target of rapamycin signaling. Additionally, we showed that PA interacted with and positively regulated sphingosine kinase 1. Taken together, our study not only generates a rich interactome resource for further characterizing the human PLD-PA lipid signaling but also connects this important metabolic pathway with numerous biological processes.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

In silico reconstitution of DNA replication. Lessons from single-molecule imaging and cryo-tomography applied to single-particle cryo-EM

DNA replication has been reconstituted in vitro with yeast proteins, and the minimal system requires the coordinated assembly of 16 distinct replication factors, consisting of 42 polypeptides. To understand the molecular interplay between these factors at the single residue level, new structural biology tools are being developed. Inspired by advances in single-molecule fluorescence imaging and cryo-tomography, novel single-particle cryo-EM experiments have been used to characterise the structural mechanism for the loading of the replicative helicase. Here, we discuss how in silico reconstitution of single-particle cryo-EM data can help describe dynamic systems that are difficult to approach with conventional three-dimensional classification tools.     

Call for biotechnological approach to degrade plastic in the era of COVID-19 pandemic

Plastic pollution is a global issue and has become a major concern since Coronavirus disease (COVID)-19. In developing nations, landfilling and illegal waste disposal are typical ways to dispose of COVID-19-infected material. These technologies worsen plastic pollution and other human and animal health problems. Plastic degrades in light and heat, generating hazardous primary and secondary micro-plastic. Certain bacteria can degrade artificial polymers using genes, enzymes, and metabolic pathways. Microorganisms including bacteria degrade petrochemical plastics slowly. High molecular weight, strong chemical bonds, and excessive hydrophobicity reduce plastic biodegradation. There is not enough study on genes, enzymes, and bacteria-plastic interactions. Synthetic biology, metabolic engineering, and bioinformatics methods have been created to biodegrade synthetic polymers. This review will focus on how microorganisms' degrading capacity can be increased using recent biotechnological techniques.     

Selective Inhibition of Cysteine-Dependent Enzymes by Bioorthogonal Tethering

A general approach for the rapid and selective inhibition of enzymes in cells using a common tool compound would be of great value for research and therapeutic development. We previously reported a chemogenetic strategy that addresses this challenge for kinases, relying on bioorthogonal tethering of a pan inhibitor to a target kinase through a genetically encoded non-canonical amino acid. However, pan inhibitors are not available for many enzyme classes. Here, we expand the scope of the chemogenetic strategy to cysteine-dependent enzymes by bioorthogonal tethering of electrophilic warheads. For proof of concept, selective inhibition of two E2 ubiquitin-conjugating enzymes, UBE2L3 and UBE2D1, was demonstrated in biochemical assays. Further development and optimization of this approach should enable its use in cells as well as with other cysteine-dependent enzymes, facilitating the investigation of their cellular function and validation as therapeutic targets.     

SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae

SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3′ non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were ～10 nt, but it was required when flaps were ～500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from ～20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about ～20 nt and is completely required at ～50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the ～20–50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.     

Intradimeric Walker A ATPases: Conserved Features of A Functionally Diverse Family

Nucleoside-triphosphate hydrolases (NTPases) are a diverse, but essential group of enzymes found in all living organisms. NTPases that have a G-X-X-X-X-G-K-[S/T] consensus sequence (where X is any amino acid), known as the Walker A or P-loop motif, constitute a superfamily of P-loop NTPases. A subset of ATPases within this superfamily contains a modified Walker A motif, X-K-G-G-X-G-K-[S/T], wherein the first invariant lysine residue is essential to stimulate nucleotide hydrolysis. Although the proteins in this subset have vastly differing functions, ranging from electron transport during nitrogen fixation to targeting of integral membrane proteins to their correct membranes, they have evolved from a shared ancestor and have thus retained common structural features that affect their functions. These commonalities have only been disparately characterized in the context of their individual proteins systems, but have not been generally annotated as features that unite the members of this family. In this review, we report an analysis based on the sequences, structures, and functions of several members in this family that highlight their remarkable similarities. A principal feature of these proteins is their dependence on homodimerization. Since their functionalities are heavily influenced by changes that happen in conserved elements at the dimer interface, we refer to the members of this subclass as intradimeric Walker A ATPases.