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1.
Nat Commun ; 12(1): 4843, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376693

RESUMO

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Repetições de Microssatélites/genética , Proteínas de Ligação a Telômeros/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Reparo do DNA por Junção de Extremidades , DNA Polimerase I/genética , DNA Primase/genética , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
2.
Nat Commun ; 12(1): 4020, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188055

RESUMO

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3'-terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3'-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.


Assuntos
Pareamento de Bases/genética , Dano ao DNA/genética , DNA Primase/metabolismo , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/análogos & derivados , Enzimas Multifuncionais/metabolismo , Guanina/química , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Estresse Oxidativo/genética , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo
3.
Nat Commun ; 12(1): 3690, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140468

RESUMO

CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.


Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroidetes/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/genética , Bacteroidetes/enzimologia , Biologia Computacional , DNA Primase/genética , Primers do DNA/biossíntese , DNA Polimerase Dirigida por DNA/genética , Dimerização , Escherichia coli/metabolismo , Expressão Gênica , Mutação , Filogenia , Células Procarióticas/metabolismo , Proteínas Recombinantes , Ribonucleotídeos/metabolismo
4.
Nat Commun ; 12(1): 2420, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893297

RESUMO

Bacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host's restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering.


Assuntos
2-Aminopurina/análogos & derivados , Adenina/química , Bacteriófagos/genética , Cianobactérias/virologia , DNA Viral/química , Synechococcus/virologia , 2-Aminopurina/química , 2-Aminopurina/metabolismo , Adenina/metabolismo , Bacteriófagos/metabolismo , Sítios de Ligação/genética , Biocatálise , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
Nucleic Acids Res ; 49(8): 4599-4612, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33849056

RESUMO

The eukaryotic replisome is comprised of three family-B DNA polymerases (Polα, δ and ϵ). Polα forms a stable complex with primase to synthesize short RNA-DNA primers, which are subsequently elongated by Polδ and Polϵ in concert with proliferating cell nuclear antigen (PCNA). In some species of archaea, family-D DNA polymerase (PolD) is the only DNA polymerase essential for cell viability, raising the question of how it alone conducts the bulk of DNA synthesis. We used a hyperthermophilic archaeon, Thermococcus kodakarensis, to demonstrate that PolD connects primase to the archaeal replisome before interacting with PCNA. Whereas PolD stably connects primase to GINS, a component of CMG helicase, cryo-EM analysis indicated a highly flexible PolD-primase complex. A conserved hydrophobic motif at the C-terminus of the DP2 subunit of PolD, a PIP (PCNA-Interacting Peptide) motif, was critical for the interaction with primase. The dissociation of primase was induced by DNA-dependent binding of PCNA to PolD. Point mutations in the alternative PIP-motif of DP2 abrogated the molecular switching that converts the archaeal replicase from de novo to processive synthesis mode.


Assuntos
Proteínas Arqueais/metabolismo , DNA Helicases/metabolismo , DNA Polimerase III/metabolismo , DNA Primase/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Thermococcus/metabolismo , Motivos de Aminoácidos , Proteínas Arqueais/química , Cromatografia em Gel , DNA Helicases/genética , DNA Polimerase III/química , DNA Primase/genética , DNA Primase/metabolismo , Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Eletroforese em Gel de Poliacrilamida Nativa , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície , Thermococcus/genética
6.
Genes (Basel) ; 12(4)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33918798

RESUMO

The mycobacterial nonhomologous end-joining pathway (NHEJ) involved in double-strand break (DSB) repair consists of the multifunctional ATP-dependent ligase LigD and the DNA bridging protein Ku. The other ATP-dependent ligases LigC and AEP-primase PrimC are considered as backup in this process. The engagement of LigD, LigC, and PrimC in the base excision repair (BER) process in mycobacteria has also been postulated. Here, we evaluated the sensitivity of Mycolicibacterium smegmatis mutants defective in the synthesis of Ku, Ku-LigD, and LigC1-LigC2-PrimC, as well as mutants deprived of all these proteins to oxidative and nitrosative stresses, with the most prominent effect observed in mutants defective in the synthesis of Ku protein. Mutants defective in the synthesis of LigD or PrimC/LigC presented a lower frequency of spontaneous mutations than the wild-type strain or the strain defective in the synthesis of Ku protein. As identified by whole-genome sequencing, the most frequent substitutions in all investigated strains were T→G and A→C. Double substitutions, as well as insertions of T or CG, were exclusively identified in the strains carrying functional Ku and LigD proteins. On the other hand, the inactivation of Ku/LigD increased the efficiency of the deletion of G in the mutant strain.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , DNA Primase/metabolismo , Ligases/metabolismo , Taxa de Mutação , Mycobacterium/genética , Estresse Oxidativo , Proteínas de Bactérias/genética , DNA Primase/genética , Ligases/genética , Mycobacterium/crescimento & desenvolvimento , Mycobacterium/metabolismo
7.
Commun Biol ; 4(1): 349, 2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731801

RESUMO

The human CST complex composed of CTC1, STN1, and TEN1 is critically involved in telomere maintenance and homeostasis. Specifically, CST terminates telomere extension by inhibiting telomerase access to the telomeric overhang and facilitates lagging strand fill in by recruiting DNA Polymerase alpha primase (Pol α-primase) to the telomeric C-strand. Here we reveal that CST has a dynamic intracellular localization that is cell cycle dependent. We report an increase in nuclear CST several hours after the initiation of DNA replication, followed by exit from the nucleus prior to mitosis. We identify amino acids of CTC1 involved in Pol α-primase binding and nuclear localization. We conclude, the CST complex does not contain a nuclear localization signal (NLS) and suggest that its nuclear localization is reliant on Pol α-primase. Hypomorphic mutations affecting CST nuclear import are associated with telomere syndromes and cancer, emphasizing the important role of this process in health.


Assuntos
Núcleo Celular/enzimologia , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Homeostase do Telômero , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Núcleo Celular/genética , DNA Polimerase I/genética , DNA Primase/genética , Replicação do DNA , Células HEK293 , Humanos , Mitose , Complexos Multiproteicos , Mutação , Ligação Proteica , Telômero/genética , Proteínas de Ligação a Telômeros/genética
8.
Nucleic Acids Res ; 49(4): 2179-2191, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33533925

RESUMO

Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.


Assuntos
Arginina/química , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , DNA/biossíntese , Enzimas Multifuncionais/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Motivos de Aminoácidos , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleotídeos/química , Desoxirribonucleotídeos/metabolismo , Modelos Moleculares , Enzimas Multifuncionais/metabolismo , Ligação Proteica
9.
DNA Repair (Amst) ; 100: 103048, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33571927

RESUMO

Human primase and DNA polymerase PrimPol re-starts stalled replication forks by repriming downstream DNA lesions and protects cells against DNA damage. Structure of the catalytic core of PrimPol with DNA primer, template and incoming dATP was solved but the mechanisms of DNA polymerase and primase activities of PrimPol are not fully understood. In this work, using site-directed mutagenesis we biochemically analyzed the role of active site residues Arg47 and Arg76 contacting DNA template in DNA polymerase and primase activities of PrimPol. The substitution R47A diminished the DNA polymerase and primase activities of PrimPol whereas the single amino acid substitution R76A caused almost complete loss of catalytic activities. Both amino acid substitutions affected the spectrum of dNMPs incorporation on undamaged DNA templates and opposite 8-oxoguanine. Finally, substitutions of the Arg47 and Arg76 residues attenuated the formation of the stable PrimPol:DNA complex in the presence of ATP/dNTPs. Together, these findings suggest a key role of the Arg47 and Arg76 in DNA synthesis by PrimPol.


Assuntos
Domínio Catalítico , Dano ao DNA , DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Guanina/análogos & derivados , Enzimas Multifuncionais/metabolismo , Sequência de Aminoácidos , DNA Primase/química , DNA Polimerase Dirigida por DNA/química , Guanina/metabolismo , Humanos , Enzimas Multifuncionais/química , Alinhamento de Sequência
10.
mSphere ; 6(1)2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504660

RESUMO

Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single-molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and lagging-strand polymerase DnaE in the model bacterium Bacillus subtilis, we show that proteins react differently to stress conditions in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after the arrest of polymerase, but binds less frequently after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone (p)ppGpp due to stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behavior of DnaG and DnaE suggests that both proteins are recruited independently to the forks rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension.IMPORTANCE All cells need to adjust DNA replication, which is achieved by a well-orchestrated multiprotein complex, in response to changes in physiological and environmental conditions. For replication forks, it is extremely challenging to meet with conditions where amino acids are rapidly depleted from cells, called the stringent response, to deal with the inhibition of one of the centrally involved proteins or with DNA modifications that arrest the progression of forks. By tracking helicase (DnaC), primase (DnaG), and polymerase (DnaE), central proteins of Bacillus subtilis replication forks, at a single molecule level in real time, we found that interactions of the three proteins with replication forks change in different manners under different stress conditions, revealing an intriguing plasticity of replication forks in dealing with replication obstacles. We have devised a new tool to determine rates of exchange between static movement (binding to a much larger complex) and free diffusion, showing that during stringent response, all proteins have highly increased exchange rates, slowing down overall replication, while inactivation of polymerase or replication roadblocks leaves forks largely intact, allowing rapid restart once obstacles are removed.


Assuntos
Bacillus subtilis/genética , Replicação do DNA , Imagem Individual de Molécula/métodos , DNA Helicases/metabolismo , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Microscopia de Fluorescência
11.
Mol Cell ; 81(4): 756-766.e8, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33472056

RESUMO

Bacillus subtilis structural maintenance of chromosomes (SMC) complexes are topologically loaded at centromeric sites adjacent to the replication origin by the partitioning protein ParB. These ring-shaped ATPases then translocate down the left and right chromosome arms while tethering them together. Here, we show that the site-specific recombinase XerD, which resolves chromosome dimers, is required to unload SMC tethers when they reach the terminus. We identify XerD-specific binding sites in the terminus region and show that they dictate the site of unloading in a manner that depends on XerD but not its catalytic residue, its partner protein XerC, or the recombination site dif. Finally, we provide evidence that ParB and XerD homologs perform similar functions in Staphylococcus aureus. Thus, two broadly conserved factors that act at the origin and terminus have second functions in loading and unloading SMC complexes that travel between them.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/metabolismo , Integrases/metabolismo , Staphylococcus aureus/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , DNA Primase/genética , DNA Primase/metabolismo , Integrases/genética , Staphylococcus aureus/genética
12.
Life Sci Alliance ; 4(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33199509

RESUMO

The mechanisms underlying multipartite genome maintenance and its functional significance in extraordinary radioresistance of Deinococcus radiodurans are not well understood. The sequences upstream to parAB operons in chrII (cisII) and MP (cisMP) could stabilize an otherwise, non-replicative colE1 plasmid, in D. radiodurans DnaA and cognate ParB proteins bound specifically with cisII and cisMP elements. The ΔcisII and ΔcisMP cells showed the reduced copy number of cognate replicons and radioresistance as compared with wild type. Fluorescent reporter-operator system inserted in chrI, chrII, and MP in wild type and cisII mutants showed the presence of all three replicons in wild-type cells. Although chrI was present in all the ΔcisII and ΔcisMP cells, nearly half of these cells had chrII and MP, respectively, and the other half had the reduced number of foci representing these replications. These results suggested that cisII and cisMP elements contain both origin of replication and parS-like functions and the secondary genome replicons (chrII and MP) are maintained independent of chrI and have roles in radioresistance of D. radiodurans.


Assuntos
Deinococcus/genética , Óperon , Origem de Replicação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Clonagem Molecular/métodos , DNA Primase/genética , DNA Primase/metabolismo , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deinococcus/efeitos da radiação , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Inativação de Genes , Plasmídeos/genética , Plasmídeos/metabolismo
13.
Int J Mol Sci ; 21(23)2020 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-33261049

RESUMO

Human PrimPol is a unique enzyme possessing DNA/RNA primase and DNA polymerase activities. In this work, we demonstrated that PrimPol efficiently fills a 5-nt gap and possesses the conditional strand displacement activity stimulated by Mn2+ ions and accessory replicative proteins RPA and PolDIP2. The DNA displacement activity of PrimPol was found to be more efficient than the RNA displacement activity and FEN1 processed the 5'-DNA flaps generated by PrimPol in vitro.


Assuntos
DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Endonucleases Flap/metabolismo , Humanos , Manganês/farmacologia , Proteínas Nucleares/metabolismo , RNA/metabolismo , Proteína de Replicação A/metabolismo , Especificidade por Substrato/efeitos dos fármacos
14.
Nat Commun ; 11(1): 5863, 2020 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-33203852

RESUMO

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.


Assuntos
Adutos de DNA/genética , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Homóloga/fisiologia , Enzimas Multifuncionais/metabolismo , 4-Nitroquinolina-1-Óxido/toxicidade , 7,8-Di-Hidro-7,8-Di-Hidroxibenzo(a)pireno 9,10-óxido/metabolismo , Benzo(a)Antracenos/administração & dosagem , Benzo(a)Antracenos/toxicidade , Linhagem Celular , Adutos de DNA/metabolismo , DNA Primase/genética , DNA de Cadeia Simples , DNA Polimerase Dirigida por DNA/genética , Humanos , Enzimas Multifuncionais/genética , Quinolonas/toxicidade , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Imagem Individual de Molécula , Troca de Cromátide Irmã
15.
J Biol Chem ; 295(50): 17298-17309, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33055234

RESUMO

The faithful segregation, or "partition," of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parA R351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a "super-repressor." In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.


Assuntos
Bacteriófago P1/metabolismo , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Bacteriófago P1/química , Bacteriófago P1/genética , Cromossomos Bacterianos/química , Cromossomos Bacterianos/genética , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/virologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação de Sentido Incorreto , Regiões Operadoras Genéticas , Proteínas Virais/química , Proteínas Virais/genética
16.
Elife ; 92020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32945259

RESUMO

Mobile genetic elements (MGEs) are a rich source of new enzymes, and conversely, understanding the activities of MGE-encoded proteins can elucidate MGE function. Here, we biochemically characterize three proteins encoded by a conserved operon carried by the Staphylococcal Cassette Chromosome (SCCmec), an MGE that confers methicillin resistance to Staphylococcus aureus, creating MRSA strains. The first of these proteins, CCPol, is an active A-family DNA polymerase. The middle protein, MP, binds tightly to CCPol and confers upon it the ability to synthesize DNA primers de novo. The CCPol-MP complex is therefore a unique primase-polymerase enzyme unrelated to either known primase family. The third protein, Cch2, is a 3'-to-5' helicase. Cch2 additionally binds specifically to a dsDNA sequence downstream of its gene that is also a preferred initiation site for priming by CCPol-MP. Taken together, our results suggest that this is a functional replication module for SCCmec.


Assuntos
Proteínas de Bactérias/genética , DNA Helicases/genética , DNA Primase/genética , Resistência a Meticilina/genética , Staphylococcus aureus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , DNA Primase/química , DNA Primase/metabolismo , Alinhamento de Sequência , Staphylococcus aureus/enzimologia
17.
Nat Commun ; 11(1): 4196, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826907

RESUMO

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol's function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC's gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC's gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols.


Assuntos
Proteínas de Bactérias/química , DNA Primase/química , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA/química , Mycobacterium/genética , Mycobacterium/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
18.
Environ Microbiol ; 22(11): 4647-4657, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32830367

RESUMO

Primase-polymerases (Ppol) are one of the few enzymes able to start DNA synthesis on ssDNA templates. The role of Thermus thermophilus HB27 Ppol, encoded along a putative helicase (Hel) within a mobile genetic element (ICETh2), has been studied. A mutant lacking Ppol showed no effects on the replication of the element. Also, no apparent differences in the sensitivity to DNA damaging agents and other stressors or morphological changes in the mutant cells were detected. However, the mutants lacking Ppol showed an increase in two to three orders of magnitude in their transformation efficiency with plasmids and genomic DNA acquired from the environment (eDNA), independently of its origin and G + C content. In contrast, no significant differences with the wild type were detected when the cells received the DNA from other T. thermophilus partners in conjugation-like mating experiments. The similarities of this behaviour with that shown by mutants lacking the Argonaute (ThAgo) protein suggests a putative partnership Ppol-ThAgo in the DNA-DNA interference mechanism of defence, although other eDNA defence mechanisms independent of ThAgo cannot be discarded.


Assuntos
Proteínas Argonauta/genética , DNA Primase/genética , DNA Ambiental/genética , Sequências Repetitivas Dispersas/genética , Thermus thermophilus/genética , Composição de Bases/genética , DNA Primase/metabolismo , Replicação do DNA/genética , DNA de Cadeia Simples/metabolismo , Deleção de Genes , Plasmídeos/genética , Thermus thermophilus/metabolismo
19.
Bioorg Med Chem Lett ; 30(17): 127398, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32738995

RESUMO

In this study we developed a novel diagnostic tool for the detection of miRNA21, based on the fluorescent nucleotide morpholine naphthalimide deoxyuridine (dUrkTP). We incorporated dUrkTP into DNA through primer extension to obtain rkDNA displaying high fluorescence. We then used lambda exonuclease, a specific nuclease for 3́-monophosphate-containing DNA, to separate rkDNA from its complementary sequence. The fluorescence of the free rkDNA was quenched dramatically upon interacting with graphene oxide (GO). Our rkDNA-GO fluorescence probing system exhibited high sensitivity and selectivity for the detection of miRNA21. This inexpensive probing system, employing simple primer extension and exonuclease degradation, required only 30 min to detect its target miRNA. This strategy appears suitable for the detection of diverse types of miRNA.


Assuntos
Desoxiuridina/química , Grafite/química , MicroRNAs/análise , Espectrometria de Fluorescência , DNA Primase/metabolismo , Desoxiuridina/síntese química , Desoxiuridina/metabolismo , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Morfolinas/química , Naftalimidas/química , Técnicas de Amplificação de Ácido Nucleico
20.
J Struct Biol ; 211(2): 107548, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32535228

RESUMO

We report the crystal structure of the SARS-CoV-2 putative primase composed of the nsp7 and nsp8 proteins. We observed a dimer of dimers (2:2 nsp7-nsp8) in the crystallographic asymmetric unit. The structure revealed a fold with a helical core of the heterotetramer formed by both nsp7 and nsp8 that is flanked with two symmetry-related nsp8 ß-sheet subdomains. It was also revealed that two hydrophobic interfaces one of approx. 1340 Å2 connects the nsp7 to nsp8 and a second one of approx. 950 Å2 connects the dimers and form the observed heterotetramer. Interestingly, analysis of the surface electrostatic potential revealed a putative RNA binding site that is formed only within the heterotetramer.


Assuntos
Betacoronavirus/química , DNA Primase/química , Proteínas não Estruturais Virais/química , Sítios de Ligação , RNA-Polimerase RNA-Dependente de Coronavírus , Cristalografia por Raios X , DNA Primase/metabolismo , Modelos Moleculares , Complexos Multiproteicos , Conformação Proteica , Multimerização Proteica , RNA/metabolismo , SARS-CoV-2 , Proteínas não Estruturais Virais/metabolismo
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