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Viruses Sep 2023Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated...
Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated over twenty years ago, the Cooper strain, used in this study, was sequenced as recently as 2012 and is currently said to encode 72 unique proteins. However, tandem mass spectrometry has identified several peptides produced during active infection that align with the BoHV-1 genome in unannotated regions. One of these abundant peptides, "ORF M", aligned antisense to the DNA helicase/primase protein U5. This study characterizes the novel transcript and its protein product and provides evidence to support the existence of homolog protein-coding genes in other Herpesviruses.
Topics: Animals; Cattle; Herpesvirus 1, Bovine; Base Sequence; Simplexvirus; DNA Primase; Peptides; Herpesviridae Infections
PubMed: 37896756
DOI: 10.3390/v15101977 -
Microbiology Spectrum Dec 2023Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the...
Eukaryotic DNA replication is a highly regulated process that requires multiple replication enzymes assembled onto DNA replication origins. Due to the complexity of the cell's DNA replication machinery, most of what we know about cellular DNA replication has come from the study of viral systems. Herein, we focus our study on the assembly of the Kaposi's sarcoma-associated herpesvirus core replication complex and propose a pairwise protein-protein interaction network of six highly conserved viral core replication proteins. A detailed understanding of the interaction and assembly of the viral core replication proteins may provide opportunities to develop new strategies against viral propagation.
Topics: Herpesvirus 8, Human; Viral Proteins; DNA Replication
PubMed: 37874136
DOI: 10.1128/spectrum.02254-23 -
Antiviral Research Nov 2023Herpes is a contagious life-long infection with persistently high incidence and prevalence, causing significant disease worldwide. Current therapies have efficacy...
Herpes is a contagious life-long infection with persistently high incidence and prevalence, causing significant disease worldwide. Current therapies have efficacy against active HSV infections but no impact on the latent viral reservoir in neurons. Thus, despite treatment, disease recurs from latency and the infectious potential remains unaffected within patients. Here, efficacy of the helicase-primase inhibitor (HPI) IM-250 against chronic neuronal HSV infections utilizing two classic herpes in vivo latency/reactivation animal models (intravaginal guinea pig HSV-2 infection model and ocular mouse HSV-1 infection model) is presented. Intermittent therapy of infected animals with 4-7 cycles of IM-250 during latency silences subsequent recurrences analyzed up to 6 months. In contrast to common experience, our studies show that the latent reservoir is indeed accessible to antiviral therapy altering the latent viral reservoir such that reactivation frequency can be reduced significantly by prior IM-250 treatment. We provide evidence that antiviral treatment during HSV latency can reduce future reactivation from the latent reservoir, supporting a conceptual shift in the antiviral field, and reframing what is achievable with respect to therapy of latent neuronal HSV infections.
Topics: Humans; Animals; Mice; Guinea Pigs; DNA Primase; Virus Latency; Herpes Simplex; Herpesvirus 1, Human; Disease Models, Animal; Antiviral Agents
PubMed: 37858763
DOI: 10.1016/j.antiviral.2023.105733 -
BMC Plant Biology Oct 2023The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically...
BACKGROUND
The mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD).
RESULTS
Herein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase.
CONCLUSIONS
We conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.
Topics: Animals; DNA Primase; DNA Helicases; DNA-Directed DNA Polymerase; Arabidopsis; Mitochondria; Zinc Fingers; Ribonucleotides; DNA Replication; Bacteriophage T7
PubMed: 37803262
DOI: 10.1186/s12870-023-04477-4 -
ELife Sep 2023The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA...
The heterotrimeric Replication protein A (RPA) is the ubiquitous eukaryotic single-stranded DNA (ssDNA) binding protein and participates in nearly all aspects of DNA metabolism, especially DNA damage response. The N-terminal OB domain of the RPA70 subunit (RPA70N) is a major protein-protein interaction element for RPA and binds to more than 20 partner proteins. Previous crystallography studies of RPA70N with p53, DNA2 and PrimPol fragments revealed that RPA70N binds to amphipathic peptides that mimic ssDNA. NMR chemical-shift studies also provided valuable information on the interaction of RPA70N residues with target sequences. However, it is still unclear how RPA70N recognizes and distinguishes such a diverse group of target proteins. Here, we present high-resolution crystal structures of RPA70N in complex with peptides from eight DNA damage response proteins. The structures show that, in addition to the ssDNA mimicry mode of interaction, RPA70N employs multiple ways to bind its partners. Our results advance the mechanistic understanding of RPA70N-mediated recruitment of DNA damage response proteins.
Topics: Humans; Crystallography; DNA Damage; DNA Primase; DNA, Single-Stranded; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Eukaryota; Multifunctional Enzymes; Replication Protein A
PubMed: 37668474
DOI: 10.7554/eLife.81639 -
Nature Communications Aug 2023Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood....
Activation of the KRAS oncogene is a source of replication stress, but how this stress is generated and how it is tolerated by cancer cells remain poorly understood. Here we show that induction of KRAS expression in untransformed cells triggers H3K27me3 and HP1-associated chromatin compaction in an RNA transcription dependent manner, resulting in replication fork slowing and cell death. Furthermore, elevated ATR expression is necessary and sufficient for tolerance of KRAS-induced replication stress to expand replication stress-tolerant cells (RSTCs). PrimPol is phosphorylated at Ser255, a potential Chk1 substrate site, under KRAS-induced replication stress and promotes repriming to maintain fork progression and cell survival in an ATR/Chk1-dependent manner. However, ssDNA gaps are generated at heterochromatin by PrimPol-dependent repriming, leading to genomic instability. These results reveal a role of ATR-PrimPol in enabling precancerous cells to survive KRAS-induced replication stress and expand clonally with accumulation of genomic instability.
Topics: Humans; Ataxia Telangiectasia Mutated Proteins; Chromatin; DNA Primase; DNA-Directed DNA Polymerase; Genomic Instability; Heterochromatin; Multifunctional Enzymes; Proto-Oncogene Proteins p21(ras)
PubMed: 37591859
DOI: 10.1038/s41467-023-40578-2 -
Nucleic Acids Research Sep 2023African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other...
African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is available. The genome of ASFV encodes more than 170 proteins, but the structures and functions for the majority of the proteins remain elusive, which hindered our understanding on the life cycle of ASFV and the development of ASFV-specific inhibitors. Here, we report the structural and biochemical studies of the highly conserved C962R protein of ASFV, showing that C962R is a multidomain protein. The N-terminal AEP domain is responsible for the DNA polymerization activity, whereas the DNA unwinding activity is catalyzed by the central SF3 helicase domain. The middle PriCT2 and D5_N domains and the C-terminal Tail domain all contribute to the DNA unwinding activity of C962R. C962R preferentially works on forked DNA, and likely functions in Base-excision repair (BER) or other repair pathway in ASFV. Although it is not essential for the replication of ASFV, C962R can serve as a model and provide mechanistic insight into the replicative primase proteins from many other species, such as nitratiruptor phage NrS-1, vaccinia virus (VACV) and other viruses.
Topics: Animals; African Swine Fever; African Swine Fever Virus; Swine; Viral Proteins; DNA Topoisomerases, Type I; DNA Replication
PubMed: 37587714
DOI: 10.1093/nar/gkad677 -
BioRxiv : the Preprint Server For... Oct 2023Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human...
Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), unique to ALT cells, are considered potential precursors of C-circles, their generation process remains undefined. Here, we introduce a highly sensitive method to detect single stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear C-rich ssDNA accumulation may indeed precede C-circle formation. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a new model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.
PubMed: 37577643
DOI: 10.1101/2023.07.31.551186 -
BioRxiv : the Preprint Server For... Aug 2023DNA replication in eukaryotes relies on the synthesis of a ~30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase...
DNA replication in eukaryotes relies on the synthesis of a ~30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ~20 nucleotides of DNA. Here we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
PubMed: 37577606
DOI: 10.1101/2023.08.01.551538