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1. Translation-Independent Roles of RNA Secondary Structures within the Replication Protein Coding Region of Turnip Crinkle Virus

   https://doi.org/10.3390/v12030350  

   Sun, Rong; Zhang, Shaoyan; Zheng, Limin; Qu, Feng (March 2020, Viruses)

   RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication. 

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2. The role of fork stalling and DNA structures in causing chromosome fragility

   https://doi.org/10.1002/gcc.22721  

   Kaushal, Simran; Freudenreich, Catherine H. (January 2019, Genes, Chromosomes and Cancer)

   Abstract Alternative non‐B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single‐stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT‐rich and break under replication stress, yet the molecular basis for their fragility is still being investigated. Over the past several years, new evidence has linked both the formation of secondary structures and transcription to fork stalling and fragility of CFSs. How these two events may synergize to cause fragility and the role of nuclease cleavage at secondary structures in rare and CFSs are discussed here. We also highlight evidence for a new hypothesis that secondary structures at CFSs not only initiate fragility but also inhibit healing, resulting in their characteristic appearance. 

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3. Structural basis for a highly conserved RNA-mediated enteroviral genome replication

   https://doi.org/10.1093/nar/gkae627  

   Das, Naba_Krishna; Vogt, Jeff; Patel, Alisha; Banna, Hasan_Al; Koirala, Deepak (July 2024, Nucleic Acids Research)

   Abstract Enteroviruses contain conserved RNA structures at the extreme 5′ end of their genomes that recruit essential proteins 3CD and PCBP2 to promote genome replication. However, the high-resolution structures and mechanisms of these replication-linked RNAs (REPLRs) are limited. Here, we determined the crystal structures of the coxsackievirus B3 and rhinoviruses B14 and C15 REPLRs at 1.54, 2.2 and 2.54 Å resolution, revealing a highly conserved H-type four-way junction fold with co-axially stacked sA-sD and sB-sC helices that are stabilized by a long-range A•C•U base-triple. Such conserved features observed in the crystal structures also allowed us to predict the models of several other enteroviral REPLRs using homology modeling, which generated models almost identical to the experimentally determined structures. Moreover, our structure-guided binding studies with recombinantly purified full-length human PCBP2 showed that two previously proposed binding sites, the sB-loop and 3′ spacer, reside proximally and bind a single PCBP2. Additionally, the DNA oligos complementary to the 3′ spacer, the high-affinity PCBP2 binding site, abrogated its interactions with enteroviral REPLRs, suggesting the critical roles of this single-stranded region in recruiting PCBP2 for enteroviral genome replication and illuminating the promising prospects of developing therapeutics against enteroviral infections targeting this replication platform. 

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4. High-Resolution Structure of the Nuclease Domain of the Human Parvovirus B19 Main Replication Protein NS1

   https://doi.org/10.1128/jvi.02164-21  

   Sanchez, Jonathan L.; Ghadirian, Niloofar; Horton, Nancy C. (May 2022, Journal of Virology)

   Frappier, Lori (Ed.)

   ABSTRACT Two new structures of the N-terminal domain of the main replication protein, NS1, of human parvovirus B19 (B19V) are presented here. This domain (NS1-nuc) plays an important role in the “rolling hairpin” replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e., nicking) single-stranded DNA at a nearby site known as the terminal resolution site (trs). The three-dimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain; however, it is shown here at a significantly higher resolution (2.4 Å). Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented that predict residues important for DNA cleavage and for sequence-specific recognition at the viral origin of replication. IMPORTANCE The high-resolution structure of the DNA binding and cleavage domain of the main replicative protein, NS1, from the human-pathogenic virus human parvovirus B19 is presented here. Included also are predictions of how the protein recognizes important sequences in the viral DNA which are required for viral replication. These predictions can be used to further investigate the function of this protein, as well as to predict the effects on viral viability due to mutations in the viral protein and viral DNA sequences. Finally, the high-resolution structure facilitates structure-guided drug design efforts to develop antiviral compounds against this important human pathogen. 

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5. DNA Polymerase Delta Exhibits Altered Catalytic Properties on Lysine Acetylation

   https://doi.org/10.3390/genes14040774  

   Njeri, Catherine; Pepenella, Sharon; Battapadi, Tripthi; Bambara, Robert A; Balakrishnan, Lata (April 2023, Genes)

   DNA polymerase delta is the primary polymerase that is involved in undamaged nuclear lagging strand DNA replication. Our mass-spectroscopic analysis has revealed that the human DNA polymerase δ is acetylated on subunits p125, p68, and p12. Using substrates that simulate Okazaki fragment intermediates, we studied alterations in the catalytic properties of acetylated polymerase and compared it to the unmodified form. The current data show that the acetylated form of human pol δ displays a higher polymerization activity compared to the unmodified form of the enzyme. Additionally, acetylation enhances the ability of the polymerase to resolve complex structures such as G-quadruplexes and other secondary structures that might be present on the template strand. More importantly, the ability of pol δ to displace a downstream DNA fragment is enhanced upon acetylation. Our current results suggest that acetylation has a profound effect on the activity of pol δ and supports the hypothesis that acetylation may promote higher-fidelity DNA replication. 

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