Newly identified HMO-2011-type phages reveal genomic diversity and biogeographic distributions of this marine viral group

Viruses play critical roles in influencing biogeochemical cycles and adjusting host mortality, population structure, physiology, and evolution in the ocean. Marine viral communities are composed of numerous genetically distinct subfamily/genus-level viral groups. Among currently identified viral groups, the HMO-2011-type group is known to be dominant and broadly distributed. However, only four HMO-2011-type cultivated representatives that infect marine SAR116 and Roseobacter strains have been reported to date, and the genetic diversity, potential hosts, and ecology of this group remain poorly elucidated. Here, we present the genomes of seven HMO-2011-type phages that were isolated using four Roseobacter strains and one SAR11 strain, as well as additional 207 HMO-2011-type metagenomic viral genomes (MVGs) identified from various marine viromes. Phylogenomic and shared-gene analyses revealed that the HMO-2011-type group is a subfamily-level group comprising at least 10 discernible genus-level subgroups. Moreover, >2000 HMO-2011-type DNA polymerase sequences were identified, and the DNA polymerase phylogeny also revealed that the HMO-2011-type group contains diverse subgroups and is globally distributed. Metagenomic read-mapping results further showed that most HMO-2011-type phages are prevalent in global oceans and display distinct geographic distributions, with the distribution of most HMO-2011-type phages being associated with temperature. Lastly, we found that members in subgroup IX, represented by pelagiphage HTVC033P, were among the most abundant HMO-2011-type phages, which implies that SAR11 bacteria are crucial hosts for this viral group. In summary, our findings substantially expand current knowledge regarding the phylogenetic diversity, evolution, and distribution of HMO-2011-type phages, highlighting HMO-2011-type phages as major ecological agents that can infect certain key bacterial groups.

A simple bypass assay for DNA polymerases shows hypermutating variants associated with cancer show mechanistic differences in vitro

Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, to genome instability and its associated diseases. Recently the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer-associated polymerase variants and the recognition that a subgroup of these variants have a hypermutation phenotype in tumours. We have developed a bypass assay to rapidly determine the tendency of a polymerase to misincorporate in vitro. We have used the assay to compare misincorporation by wild-type, exonuclease defective and two hypermutating DNA polymerase e variants, P286R and V411L. The assay clearly distinguished between the misincorporation rates of wild type, exonuclease dead and P286R polymerases. However, the V411L polymerase showed different misincorporation characteristics to P286R, suggesting that these variants cause hypermutation by different mechanisms. Using this assay misincorporation opposite a templated C nucleotide was consistently higher than for other nucleotides, and this caused predominantly C to T transitions. This is consistent with the observation that C to T transitions are commonly seen in POLE mutant tumours.

Modified Cytosines versus Cytosine in a DNA Polymerase: Retrieving Thermodynamic and Kinetic Constants at the Single Molecule Level

DNA methylation plays key roles in various areas, such as gene expression, regulation, epigenetics, and cancers. Since 5-methylcytosine (5mC) is commonly present in methylated DNA, characterizing the binding kinetics and...

Introducing a New Bond-Forming Activity in an Archaeal DNA Polymerase by Structure-Guided Enzyme Redesign

DNA polymerases have evolved to feature a highly conserved activity across the tree of life: formation of, without exception, phosphodiester linkages that create the repeating sugar-phosphate backbone of DNA. Can this linkage selectivity observed in nature be overcome by design to produce non-natural nucleic acids? Here, we report that structure-guided redesign of an archaeal DNA polymerase (9°N) enables a new polymerase activity that is undetectable in the wild type enzyme: catalyzing the formation of N3′→P5′ phosphoramidate linkages in the presence of 3′-amino-2′,3′-dideoxynucleoside 5′-triphosphate (3′-NH2-ddNTP) building blocks. Replacing a highly conserved metal-binding aspartate in the 9°N active site (Asp-404) with asparagine was key to the emergence of this unnatural enzyme activity. Molecular dynamics simulations provided insights into how a single substitution could enhance the productive positioning of the 3′-amino nucleophile in the active site. Further remodeling of the protein-nucleic acid interface with substitutions in the finger subdomain led to a quadruple-mutant variant (9°N-NRQS) that incorporated 3′-NH2-ddNTPs into a 3′-amino-primer on various DNA templates. This work presents the first example of an active-site substitution of a metal-binding residue that leads to a novel activity in a DNA polymerase, and sheds light on the molecular basis of substrate fidelity and latent promiscuity in enzymes.

Monovalent Copper Ions Inhibit Enzymatic Systems

The effect of monovalent copper ions on enzymatic systems has hardly been studied to date; this is due to the low stability of monovalent copper ions in aqueous solutions, which led to the assumption that their concentration is negligible in biological systems. However, in an anaerobic atmosphere, and in the presence of a ligand that stabilizes the monovalent copper ions over the divalent copper ions, high and stable concentrations of monovalent copper ions can be reached. Moreover, the cell cytoplasm has a substantial concentration of potential stabilizers that can explain significant concentrations of monovalent copper ions in the cytoplasm. This study demonstrates the effect of monovalent and divalent copper ions on DNA polymerase, ligaseT4 DNA, the restriction enzymes EcoP15I and EcoR I, acid phosphatase, and α and βamylase enzymes. These systems were chosen because they can be monitored under conditions necessary for maintaining a stable concentration of monovalent copper ions, and since they exhibit a wide range of dependency on ATP. Previous studies indicated that ATP interacts with monovalent and divalent copper ions and stabilizing monovalent copper ions over divalent copper ions. The results showed that monovalent copper ions dramatically inhibit DNA polymerase and acid phosphatase, inhibit ligaseT4 DNA and the restriction enzyme EcoP15I, moderately inhibit α and β amylase, and have no effect on the restriction enzyme EcoR I. From the results presented in this work, it can be concluded that the mechanism is not one of oxidative stress, even though monovalent copper ions generate reactive oxygen species (ROS). Molecular oxygen in the medium, which is supposed to increase the oxidative stress, impairs the inhibitory effect of monovalent and divalent copper ions, and the kinetics of the inhibition is not suitable for the ROS mechanism.ATP forms a complex with copper ions (di and monovalent ions, where the latter is more stable) in which the metal ion is bound both to the nitrogen base and to the oxygen charged on the phosphate groups, forming an unusually distorted complex. The results of this study indicate that these complexes have the ability to inhibit enzymatic systems that are dependent on ATP.This finding can provide an explanation for the strong antimicrobial activity of monovalent copper ions, suggesting that rapid and lethal metabolic damage is the main mechanism of monovalent copper ions’ antimicrobial effect.

Structure of telomerase-bound CST with Polymerase α-Primase

Telomeres are the physical ends of linear chromosomes, composed of short repeating sequences (e.g. TTGGGG in Tetrahymena for the G-strand) of double-stranded DNA with a single-strand 3'-overhang of the G-strand and a group of proteins called shelterin. Among these, TPP1 and POT1 associate with the 3'-overhang, with POT1 binding the G-strand and TPP1 recruiting telomerase via interaction with telomerase reverse transcriptase (TERT). The ends of the telomeric DNA are replicated and maintained by telomerase, for the G-strand, and subsequently DNA Polymerase α-Primase (PolαPrim), for the C-strand. PolαPrim is stimulated by CTC1-STN1-TEN1 (CST), but the structural basis of both PolαPrim and CST recruitment to telomere ends remains unknown. Here we report cryo-EM structures of Tetrahymena CST in the context of telomerase holoenzyme, both in the absence and presence of PolαPrim, as well as of PolαPrim alone. Ctc1 binds telomerase subunit p50, a TPP1 ortholog, on a flexible Ctc1 binding motif unveiled jointly by cryo-EM and NMR spectroscopy. PolαPrim subunits are arranged in a catalytically competent conformation, in contrast to previously reported autoinhibited conformation. Polymerase POLA1 binds Ctc1 and Stn1, and its interface with Ctc1 forms an entry port for G-strand DNA to the POLA1 active site. Together, we obtained a snapshot of four key players required for telomeric DNA synthesis in a single complex-telomerase core RNP, p50/TPP1, CST and PolαPrim-that provides unprecedented insights into CST and PolαPrim recruitment and handoff between G-strand and C-strand synthesis.

A sensitized genetic screen to identify regulators of C. elegans germline stem cells

GLP-1/Notch signaling and a downstream RNA regulatory network maintain germline stem cells (GSCs) in Caenorhabditis elegans. In mutants lacking the GLP-1 receptor, all GSCs enter the meiotic cell cycle precociously and differentiate into sperm. This dramatic GSC defect is called the “Glp” phenotype. The lst-1 and sygl-1 genes are direct targets of Notch transcriptional activation and functionally redundant. Whereas single lst-1 and sygl-1 mutants are fertile, lst-1 sygl-1 double mutants are sterile with a Glp phenotype. We set out to identify genes that function redundantly with either lst-1 or sygl-1 to maintain GSCs. To this end, we conducted forward genetic screens for mutants with a Glp phenotype in genetic backgrounds lacking functional copies of either lst-1 or sygl-1. The screens generated nine glp-1 alleles, two lst-1 alleles, and one allele of pole-1, which encodes the catalytic subunit of DNA polymerase ε. Three glp-1 alleles reside in Ankyrin (ANK) repeats not previously mutated. pole-1 single mutants have a low penetrance Glp phenotype that is enhanced by loss of sygl-1. Thus, the screen uncovered one locus that interacts genetically with sygl-1 and generated useful mutations for further studies of GSC regulation.