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Journal of Basic Microbiology Jan 2024The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several...
The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several types of associated TFs having been reported. SLCG_2919, a tetracycline repressor (TetR)-type regulator, was the first TF to be characterized outside the lincomycin biosynthetic cluster to directly suppress the lincomycin biosynthesis in S. lincolnensis. In this study, improved genomic systematic evolution of ligands by exponential enrichment (gSELEX), an in vitro technique, was adopted to capture additional SLCG_2919-targeted sequences harboring the promoter regions of SLCG_6675, SLCG_4123-4124, SLCG_6579, and SLCG_0139-0140. The four DNA fragments were confirmed by electrophoretic mobility shift assays (EMSAs). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) showed that the corresponding target genes SLCG_6675 (anthranilate synthase), SLCG_0139 (LysR family transcriptional regulator), SLCG_0140 (beta-lactamase), SLCG_6579 (cytochrome P450), SLCG_4123 (bifunctional DNA primase/polymerase), and SLCG_4124 (magnesium or magnesium-dependent protein phosphatase) in ΔSLCGL_2919 were differentially increased by 3.3-, 4.2-, 3.2-, 2.5-, 4.6-, and 2.2-fold relative to those in the parental strain S. lincolnensis LCGL. Furthermore, the individual inactivation of these target genes in LCGL reduced the lincomycin yield to varying degrees. This investigation expands on the known DNA targets of SLCG_2919 to control lincomycin production and lays the foundation for improving industrial lincomycin yields via genetic engineering of this regulatory network.
Topics: Magnesium; Bacterial Proteins; Anti-Bacterial Agents; Lincomycin; Transcription Factors; Tetracycline; DNA; Gene Expression Regulation, Bacterial; Streptomyces
PubMed: 37562983
DOI: 10.1002/jobm.202300203 -
Molecular Cell Aug 2023During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during...
During eukaryotic DNA replication, Pol α-primase generates primers at replication origins to start leading-strand synthesis and every few hundred nucleotides during discontinuous lagging-strand replication. How Pol α-primase is targeted to replication forks to prime DNA synthesis is not fully understood. Here, by determining cryoelectron microscopy (cryo-EM) structures of budding yeast and human replisomes containing Pol α-primase, we reveal a conserved mechanism for the coordination of priming by the replisome. Pol α-primase binds directly to the leading edge of the CMG (CDC45-MCM-GINS) replicative helicase via a complex interaction network. The non-catalytic PRIM2/Pri2 subunit forms two interfaces with CMG that are critical for in vitro DNA replication and yeast cell growth. These interactions position the primase catalytic subunit PRIM1/Pri1 directly above the exit channel for lagging-strand template single-stranded DNA (ssDNA), revealing why priming occurs efficiently only on the lagging-strand template and elucidating a mechanism for Pol α-primase to overcome competition from RPA to initiate primer synthesis.
Topics: Humans; DNA Primase; Cryoelectron Microscopy; DNA Replication; DNA Helicases; Saccharomyces cerevisiae; DNA, Single-Stranded
PubMed: 37506699
DOI: 10.1016/j.molcel.2023.06.035 -
Genes & Development Jul 2023It has been known for decades that telomerase extends the 3' end of linear eukaryotic chromosomes and dictates the telomeric repeat sequence based on the template in its... (Review)
Review
It has been known for decades that telomerase extends the 3' end of linear eukaryotic chromosomes and dictates the telomeric repeat sequence based on the template in its RNA. However, telomerase does not mitigate sequence loss at the 5' ends of chromosomes, which results from lagging strand DNA synthesis and nucleolytic processing. Therefore, a second enzyme is needed to keep telomeres intact: DNA polymerase α/Primase bound to Ctc1-Stn1-Ten1 (CST). CST-Polα/Primase maintains telomeres through a fill-in reaction that replenishes the lost sequences at the 5' ends. CST not only serves to maintain telomeres but also determines their length by keeping telomerase from overelongating telomeres. Here we discuss recent data on the evolution, structure, function, and recruitment of mammalian CST-Polα/Primase, highlighting the role of this complex and telomere length control in human disease.
Topics: Animals; Humans; Telomerase; DNA Primase; Telomere-Binding Proteins; Telomere; Telomere Homeostasis; DNA Replication; Mammals
PubMed: 37495394
DOI: 10.1101/gad.350479.123 -
Nature Communications Jul 2023The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is...
The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.
Topics: Bacteriophage T4; DNA Primase; DNA Helicases; DNA Replication; DNA Primers; DNA, Viral
PubMed: 37474605
DOI: 10.1038/s41467-023-40106-2 -
ACS Nano Aug 2023Direct delivery of therapeutic genes is a promising approach for treating cancers and other diseases. The current human viral vectors, however, suffer from several...
Direct delivery of therapeutic genes is a promising approach for treating cancers and other diseases. The current human viral vectors, however, suffer from several drawbacks, including poor cell-type specificity and difficult large-scale production. The M13 phage provides an alternative vehicle for gene therapy with engineerable specificity, but the low transduction efficiency seriously limits its translational application. In this work, we discovered important factors of cells and phages that greatly influence the phage transduction. The up-regulation of PrimPol or the down-regulation of DMBT1 in cells significantly enhanced the phage transduction efficiency. Furthermore, we found that the phage transduction efficiency was inversely correlated with the phage size. By carefully reconstructing the phage origin with the gene of interest, we designed "TransPhage" with a minimal length and maximal transduction efficiency. We showed that TransPhage successfully transduced the human cells with an excellent efficiency (up to 95%) comparable to or superior to that of the adeno-associated virus vectors. Moreover, we showed that TransPhage's tropism was specific to the cells that overexpress the target antigen, whereas adeno-associated viruses (AAVs) promiscuously infected many cell types. Using TransPhage as a gene therapy vehicle, we invented an NK-cell-mediated immunotherapy in which a membrane-bound fragment crystallizable region was introduced to cancer cells. We showed that the cancer cells expressing the membrane-bound fragment crystallizable (Fc) were effectively killed by CD16+ NK cells through an antibody-dependent cell-mediated cytotoxicity (ADCC)-like mechanism. In the xenograft mouse model, the administration of TransPhage carrying the membrane-bound Fc gene greatly suppressed tumor growth.
Topics: Humans; Mice; Animals; Gene Transfer Techniques; Genetic Vectors; Bacteriophage M13; Genetic Therapy; Killer Cells, Natural; Neoplasms; Calcium-Binding Proteins; DNA-Binding Proteins; Tumor Suppressor Proteins; DNA-Directed DNA Polymerase; DNA Primase; Multifunctional Enzymes
PubMed: 37466994
DOI: 10.1021/acsnano.3c01295 -
Current Opinion in Structural Biology Oct 2023Members of the primase-polymerase (Prim-Pol) superfamily are found in all domains of life and play diverse roles in genome stability, including primer synthesis during... (Review)
Review
Members of the primase-polymerase (Prim-Pol) superfamily are found in all domains of life and play diverse roles in genome stability, including primer synthesis during DNA replication, lesion repair and damage tolerance. This review focuses primarily on Prim-Pol members capable of de novo primer synthesis that have experimentally derived structural models available. We discuss the mechanism of DNA primer synthesis initiation by Prim-Pol catalytic domains, based on recent structural and functional studies. We also describe a general model for primer initiation that also includes the ancillary domains/subunits, which stimulate the initiation of primer synthesis.
Topics: DNA Primase; DNA Replication; Catalytic Domain
PubMed: 37459807
DOI: 10.1016/j.sbi.2023.102652 -
European Journal of Medical Research Jun 2023It is critical to understand the mechanisms of human cancers in order to develop the effective anti-cancer therapeutic strategies. Recent studies indicated that primase...
BACKGROUND
It is critical to understand the mechanisms of human cancers in order to develop the effective anti-cancer therapeutic strategies. Recent studies indicated that primase polymerase (PRIMPOL) is strongly associated with the development of human cancers. Nevertheless, a systematic pan-cancer analysis of PRIMPOL remains to be further clarified.
METHOD
Comprehensive multi-omics bioinformatics algorithms, such as TIMER2.0, GEPIA2.0 and cBioPortal, were utilized to evaluate the biological roles of PRIMPOL in pan-cancer, including the expression profiles, genomic alterations, prognostic values and immune regulation.
RESULTS
PRIMPOL was upregulated in glioblastoma multiforme and kidney renal clear cell carcinoma. The brain lower grade glioma patients with enhanced PRIMPOL expression displayed poor prognostic values. We also demonstrated the PRIMPOL's immunomodulating effects on pan-cancer as well as its genomic changes and methylation levels. The aberrant expression of PRIMPOL was linked to various cancer-associated pathways, including DNA damage response, DNA repair, and angiogenesis, according to single-cell sequencing and function enrichment.
CONCLUSIONS
This pan-cancer analysis offers a thorough review of the functional roles of PRIMPOL in human cancers, suggesting PRIMPOL as a potentially important biomarker for the progression and immunotherapy of various cancers.
Topics: Humans; DNA Primase; Multiomics; Prognosis; Carcinoma, Renal Cell; Kidney Neoplasms; Immunity; DNA Replication
PubMed: 37391787
DOI: 10.1186/s40001-023-01181-9 -
Bioscience Reports Jul 2023To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these... (Review)
Review
To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicative primases (eukaryotes and archaea) belong to a functionally diverse enzyme superfamily known as Primase-Polymerases (Prim-Pols), with orthologues present throughout all domains of life. Characterised by a conserved catalytic Prim-Pol domain, these enzymes have evolved various roles in DNA metabolism, including DNA replication, repair, and damage tolerance. Many of these biological roles are fundamentally underpinned by the ability of Prim-Pols to generate primers de novo. This review examines our current understanding of the catalytic mechanisms utilised by Prim-Pols to initiate primer synthesis.
Topics: DNA Primase; DNA-Directed DNA Polymerase; DNA Replication; Catalytic Domain; DNA
PubMed: 37358261
DOI: 10.1042/BSR20221986 -
Nature Communications Jun 2023The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and...
The eukaryotic polymerase α (Pol α) synthesizes an RNA-DNA hybrid primer of 20-30 nucleotides. Pol α is composed of Pol1, Pol12, Primase 1 (Pri1), and Pri2. Pol1 and Pri1 contain the DNA polymerase and RNA primase activities, respectively. It has been unclear how Pol α hands over an RNA primer from Pri1 to Pol1 for DNA primer extension, and how the primer length is defined. Here we report the cryo-EM analysis of yeast Pol α in the apo, primer initiation, primer elongation, RNA primer hand-off from Pri1 to Pol1, and DNA extension states, revealing a series of very large movements. We reveal a critical point at which Pol1-core moves to take over the 3'-end of the RNA from Pri1. DNA extension is limited by a spiral motion of Pol1-core. Since both Pri1 and Pol1-core are flexibly attached to a stable platform, primer growth produces stress that limits the primer length.
Topics: DNA Primase; DNA-Directed DNA Polymerase; DNA Replication; DNA; RNA; Saccharomyces cerevisiae; DNA Primers
PubMed: 37344454
DOI: 10.1038/s41467-023-39441-1 -
Nucleic Acids Research Aug 2023Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria....
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
Topics: Humans; DNA-Directed DNA Polymerase; DNA Primase; DNA Replication; DNA; DNA Primers; Catalysis; Multifunctional Enzymes
PubMed: 37326028
DOI: 10.1093/nar/gkad507