ResearchPad - nucleic-acid-enzymes https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Allosteric coupling between Mn<sup>2+</sup> and dsDNA controls the catalytic efficiency and fidelity of cGAS]]> https://www.researchpad.co/article/N3f2672b7-a325-4f10-9f7b-2dfec0dba4e2 Cyclic-G/AMP (cGAMP) synthase (cGAS) triggers host innate immune responses against cytosolic double-stranded (ds)DNA arising from genotoxic stress and pathogen invasion. The canonical activation mechanism of cGAS entails dsDNA-binding and dimerization. Here, we report an unexpected activation mechanism of cGAS in which Mn2+ activates monomeric cGAS without dsDNA. Importantly, the Mn2+-mediated activation positively couples with dsDNA-dependent activation in a concerted manner. Moreover, the positive coupling between Mn2+ and dsDNA length-dependent activation requires the cognate ATP/GTP substrate pair, while negative-cooperativity suppresses Mn2+ utilization by either ATP or GTP alone. Additionally, while Mn2+ accelerates the overall catalytic activity, dsDNA length-dependent dimerization specifically accelerates the cyclization of cGAMP. Together, we demonstrate how the intrinsic allostery of cGAS efficiently yet precisely tunes its activity.

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<![CDATA[Mechanistic insights from structure of <i>Mycobacterium smegmatis</i> topoisomerase I with ssDNA bound to both N- and C-terminal domains]]> https://www.researchpad.co/article/N81f0f89e-d181-4a22-8bf4-647dd2bf180d Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.

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<![CDATA[Spermidine biases the resolution of Holliday junctions by phage λ integrase]]> https://www.researchpad.co/article/5b7c3a19463d7e0e8f16b9a6

Holliday junctions are a central intermediate in diverse pathways of DNA repair and recombination. The isomerization of a junction determines the directionality of the recombination event. Previous studies have shown that the identity of the central sequence of the junction may favor one of the two isomers, in turn controlling the direction of the pathway. Here we demonstrate that, in the absence of DNA sequence-mediated isomer preference, polycations are the major contributor to biasing strand cleavage during junction resolution. In the case of wild-type phage λ excision junctions, spermidine plays the dominant role in controlling the isomerization state of the junction and increases the rate of junction resolution. Spermidine also counteracts the sequence-imposed bias on resolution. The spermidine-induced bias is seen equally on supercoiled and linear excisive recombination junction intermediates, and thus is not just an artefact of in vitro recombination conditions. The contribution of spermidine requires the presence of accessory factors, and results in the repositioning of Int's core-binding domains on junctions, perhaps due to DNA-spermidine–protein interactions, or by influencing DNA conformation in the core region. Our results lead us to propose that spermidine together with accessory factors promotes the formation of the second junction isomer. We propose that this rearrangement triggers the activation of the second pair of Int active sites necessary to resolve Holliday junctions during phage λ Int-mediated recombination.

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<![CDATA[Dynamic structural insights into the molecular mechanism of DNA unwinding by the bacteriophage T7 helicase]]> https://www.researchpad.co/article/Nc02a6159-67c3-47cb-8c4e-6edb769c83f6

Abstract

The hexametric T7 helicase (gp4) adopts a spiral lock-washer form and encircles a coil-like DNA (tracking) strand with two nucleotides bound to each subunit. However, the chemo-mechanical coupling mechanism in unwinding has yet to be elucidated. Here, we utilized nanotensioner-enhanced Förster resonance energy transfer with one nucleotide precision to investigate gp4-induced unwinding of DNA that contains an abasic lesion. We observed that the DNA unwinding activity of gp4 is hindered but not completely blocked by abasic lesions. Gp4 moves back and forth repeatedly when it encounters an abasic lesion, whereas it steps back only occasionally when it unwinds normal DNA. We further observed that gp4 translocates on the tracking strand in step sizes of one to four nucleotides. We propose that a hypothetical intermediate conformation of the gp4–DNA complex during DNA unwinding can help explain how gp4 molecules pass lesions, providing insights into the unwinding dynamics of gp4.

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<![CDATA[The critical function of the plastid rRNA methyltransferase, CMAL, in ribosome biogenesis and plant development]]> https://www.researchpad.co/article/N6f3e167d-06e3-4298-8b5d-db0efb6a4fd7

Abstract

Methylation of nucleotides in ribosomal RNAs (rRNAs) is a ubiquitous feature that occurs in all living organisms. The formation of methylated nucleotides is performed by a variety of RNA-methyltransferases. Chloroplasts of plant cells result from an endosymbiotic event and possess their own genome and ribosomes. However, enzymes responsible for rRNA methylation and the function of modified nucleotides in chloroplasts remain to be determined. Here, we identified an rRNA methyltransferase, CMAL (Chloroplast MraW-Like), in the Arabidopsis chloroplast and investigated its function. CMAL is the Arabidopsis ortholog of bacterial MraW/ RsmH proteins and accounts to the N4-methylation of C1352 in chloroplast 16S rRNA, indicating that CMAL orthologs and this methyl-modification nucleotide is conserved between bacteria and the endosymbiont-derived eukaryotic organelle. The knockout of CMAL in Arabidopsis impairs the chloroplast ribosome accumulation and accordingly reduced the efficiency of mRNA translation. Interestingly, the loss of CMAL leads not only to defects in chloroplast function, but also to abnormal leaf and root development and overall plant morphology. Further investigation showed that CMAL is involved in the plant development probably by modulating auxin derived signaling pathways. This study uncovered the important role of 16S rRNA methylation mediated by CMAL in chloroplast ribosome biogenesis and plant development.

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<![CDATA[Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme]]> https://www.researchpad.co/article/Nade28d27-54ad-44a7-b13c-c658052934da

Abstract

Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5′ exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3′-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3′ exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.

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<![CDATA[Molecular basis for t6A modification in human mitochondria]]> https://www.researchpad.co/article/N4d054181-125a-4a93-8ab8-9c07ed550c93

Abstract

N 6-Threonylcarbamoyladenosine (t6A) is a universal tRNA modification essential for translational accuracy and fidelity. In human mitochondria, YrdC synthesises an l-threonylcarbamoyl adenylate (TC-AMP) intermediate, and OSGEPL1 transfers the TC-moiety to five tRNAs, including human mitochondrial tRNAThr (hmtRNAThr). Mutation of hmtRNAs, YrdC and OSGEPL1, affecting efficient t6A modification, has been implicated in various human diseases. However, little is known about the tRNA recognition mechanism in t6A formation in human mitochondria. Herein, we showed that OSGEPL1 is a monomer and is unique in utilising C34 as an anti-determinant by studying the contributions of individual bases in the anticodon loop of hmtRNAThr to t6A modification. OSGEPL1 activity was greatly enhanced by introducing G38A in hmtRNAIle or the A28:U42 base pair in a chimeric tRNA containing the anticodon stem of hmtRNASer(AGY), suggesting that sequences of specific hmtRNAs are fine-tuned for different modification levels. Moreover, using purified OSGEPL1, we identified multiple acetylation sites, and OSGEPL1 activity was readily affected by acetylation via multiple mechanisms in vitro and in vivo. Collectively, we systematically elucidated the nucleotide requirement in the anticodon loop of hmtRNAs, and revealed mechanisms involving tRNA sequence optimisation and post-translational protein modification that determine t6A modification levels.

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<![CDATA[The internal loops in the lower stem of primary microRNA transcripts facilitate single cleavage of human Microprocessor]]> https://www.researchpad.co/article/N9c68dafe-8085-49c3-bdf9-1a203e11c530

Abstract

The human Microprocessor complex cleaves primary microRNA (miRNA) transcripts (pri-miRNAs) to initiate miRNA synthesis. Microprocessor consists of DROSHA (an RNase III enzyme), and DGCR8. DROSHA contains two RNase III domains, RIIIDa and RIIIDb, which simultaneously cleave the 3p- and 5p-strands of pri-miRNAs, respectively. In this study, we show that the internal loop located in the lower stem of numerous pri-miRNAs selectively inhibits the cleavage of Microprocessor on their 3p-strand, thereby, facilitating the single cleavage on their 5p-strand. This single cleavage does not lead to the production of miRNA but instead, it downregulates miRNA expression. We also demonstrate that by manipulating the size of the internal loop in the lower stem of pri-miRNAs, we can alter the ratio of single-cut to double-cut products resulted from the catalysis of Microprocessor, thus changing miRNA production in the in vitro pri-miRNA processing assays and in human cells. Therefore, the oscillating level of the single cleavage suggests another way of regulation of miRNA expression and offers an alternative approach to miRNA knockdown.

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<![CDATA[Mutations of R882 change flanking sequence preferences of the DNA methyltransferase DNMT3A and cellular methylation patterns]]> https://www.researchpad.co/article/N16606f40-b139-4629-a88c-130cf8eb8313

Abstract

Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3′ side of the CpG site, where they resemble DNMT3B, while 5′ flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation.

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<![CDATA[Ring-shaped replicative helicase encircles double-stranded DNA during unwinding]]> https://www.researchpad.co/article/N0acbc566-46fb-4686-99a1-418aa63bcf7d

Abstract

Ring-shaped replicative helicases are hexameric and play a key role in cellular DNA replication. Despite their importance, our understanding of the unwinding mechanism of replicative helicases is far from perfect. Bovine papillomavirus E1 is one of the best-known model systems for replicative helicases. E1 is a multifunctional initiator that senses and melts the viral origin and unwinds DNA. Here, we study the unwinding mechanism of E1 at the single-molecule level using magnetic tweezers. The result reveals that E1 as a single hexamer is a poorly processive helicase with a low unwinding rate. Tension on the DNA strands impedes unwinding, indicating that the helicase interacts strongly with both DNA strands at the junction. While investigating the interaction at a high force (26–30 pN), we discovered that E1 encircles dsDNA. By comparing with the E1 construct without a DNA binding domain, we propose two possible encircling modes of E1 during active unwinding.

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<![CDATA[AtTrm5a catalyses 1-methylguanosine and 1-methylinosine formation on tRNAs and is important for vegetative and reproductive growth in Arabidopsis thaliana]]> https://www.researchpad.co/article/5c5f1b9ad5eed0c48469b7c3

Abstract

Modified nucleosides on tRNA are critical for decoding processes and protein translation. tRNAs can be modified through 1-methylguanosine (m1G) on position 37; a function mediated by Trm5 homologs. We show that AtTRM5a (At3g56120) is a Trm5 ortholog in Arabidopsis thaliana. AtTrm5a is localized to the nucleus and its function for m1G and m1I methylation was confirmed by mutant analysis, yeast complementation, m1G nucleoside level on single tRNA, and tRNA in vitro methylation. Arabidopsis attrm5a mutants were dwarfed and had short filaments, which led to reduced seed setting. Proteomics data indicated differences in the abundance of proteins involved in photosynthesis, ribosome biogenesis, oxidative phosphorylation and calcium signalling. Levels of phytohormone auxin and jasmonate were reduced in attrm5a mutant, as well as expression levels of genes involved in flowering, shoot apex cell fate determination, and hormone synthesis and signalling. Taken together, loss-of-function of AtTrm5a impaired m1G and m1I methylation and led to aberrant protein translation, disturbed hormone homeostasis and developmental defects in Arabidopsis plants.

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<![CDATA[Site specific target binding controls RNA cleavage efficiency by the Kaposi's sarcoma-associated herpesvirus endonuclease SOX]]> https://www.researchpad.co/article/5c26b349d5eed0c48475fc4e

Abstract

A number of viruses remodel the cellular gene expression landscape by globally accelerating messenger RNA (mRNA) degradation. Unlike the mammalian basal mRNA decay enzymes, which largely target mRNA from the 5′ and 3′ end, viruses instead use endonucleases that cleave their targets internally. This is hypothesized to more rapidly inactivate mRNA while maintaining selective power, potentially though the use of a targeting motif(s). Yet, how mRNA endonuclease specificity is achieved in mammalian cells remains largely unresolved. Here, we reveal key features underlying the biochemical mechanism of target recognition and cleavage by the SOX endonuclease encoded by Kaposi's sarcoma-associated herpesvirus (KSHV). Using purified KSHV SOX protein, we reconstituted the cleavage reaction in vitro and reveal that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. The strength of SOX binding dictates cleavage efficiency, providing an explanation for the breadth of mRNA susceptibility observed in cells. Importantly, we establish that cleavage site specificity does not require additional cellular cofactors, as had been previously proposed. Thus, viral endonucleases may use a combination of RNA sequence and structure to capture a broad set of mRNA targets while still preserving selectivity.

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<![CDATA[Active site residue identity regulates cleavage preference of LAGLIDADG homing endonucleases]]> https://www.researchpad.co/article/5c26b34dd5eed0c48475fcc1

Abstract

LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications.

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<![CDATA[Human MARF1 is an endoribonuclease that interacts with the DCP1:2 decapping complex and degrades target mRNAs]]> https://www.researchpad.co/article/5c26b34bd5eed0c48475fc80

Abstract

Meiosis arrest female 1 (MARF1) is a cytoplasmic RNA binding protein that is essential for meiotic progression of mouse oocytes, in part by limiting retrotransposon expression. MARF1 is also expressed in somatic cells and tissues; however, its mechanism of action has yet to be investigated. Human MARF1 contains a NYN-like domain, two RRMs and eight LOTUS domains. Here we provide evidence that MARF1 post-transcriptionally silences targeted mRNAs. MARF1 physically interacts with the DCP1:DCP2 mRNA decapping complex but not with deadenylation machineries. Importantly, we provide a 1.7 Å resolution crystal structure of the human MARF1 NYN domain, which we demonstrate is a bona fide endoribonuclease, the activity of which is essential for the repression of MARF1-targeted mRNAs. Thus, MARF1 post-transcriptionally represses gene expression by serving as both an endoribonuclease and as a platform that recruits the DCP1:DCP2 decapping complex to targeted mRNAs.

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<![CDATA[Structural basis for recognition and repair of the 3′-phosphate by NExo, a base excision DNA repair nuclease from Neisseria meningitidis]]> https://www.researchpad.co/article/5c26b32fd5eed0c48475f707

Abstract

NExo is an enzyme from Neisseria meningitidis that is specialized in the removal of the 3′-phosphate and other 3′-lesions, which are potential blocks for DNA repair. NExo is a highly active DNA 3′-phosphatase, and although it is from the class II AP family it lacks AP endonuclease activity. In contrast, the NExo homologue NApe, lacks 3′-phosphatase activity but is an efficient AP endonuclease. These enzymes act together to protect the meningococcus from DNA damage arising mainly from oxidative stress and spontaneous base loss. In this work, we present crystal structures of the specialized 3′-phosphatase NExo bound to DNA in the presence and absence of a 3′-phosphate lesion. We have outlined the reaction mechanism of NExo, and using point mutations we bring mechanistic insights into the specificity of the 3′-phosphatase activity of NExo. Our data provide further insight into the molecular origins of plasticity in substrate recognition for this class of enzymes. From this we hypothesize that these specialized enzymes lead to enhanced efficiency and accuracy of DNA repair and that this is important for the biological niche occupied by this bacterium.

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<![CDATA[Functional characterization of a 48 kDa Trypanosoma brucei cap 2 RNA methyltransferase]]> https://www.researchpad.co/article/5b7be207463d7e7b3349a61a

Kinetoplastid mRNAs possess a unique hypermethylated cap 4 structure derived from the standard m7GpppN cap structure, with 2′-O methylations on the first four ribose sugars and additional base methylations on the first adenine and the fourth uracil. While the enzymes responsible for m7GpppN cap 0 formations has been characterized in Trypanosoma brucei, the mechanism of cap 4 methylation and the role of the hypermethylated structure remain unclear. Here, we describe the characterization of a 48 kDa T.brucei 2′-O nucleoside methyltransferase (TbCom1). Recombinant TbCom1 transfers the methyl group from S-adenosylmethionine (AdoMet) to the 2′-OH of the second nucleoside of m7GpppNpNp-RNA to form m7GpppNpNmp-RNA. TbCom1 is also capable of converting cap 1 RNA to cap 2 RNA. The methyl transfer reaction is dependent on the m7GpppN cap, as the enzyme does not form a stable interaction with GpppN-terminated RNA. Mutational analysis establishes that the TbCom1 and vaccinia virus VP39 methyltransferases share mechanistic similarities in AdoMet- and cap-recognition. Two aromatic residues, Tyr18 and Tyr187, may participate in base-stacking interactions with the guanine ring of the cap, as the removal of each of these aromatic side-chains abolishes cap-specific RNA-binding.

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<![CDATA[ Mycobacterium smegmatis DinB2 misincorporates deoxyribonucleotides and ribonucleotides during templated synthesis and lesion bypass]]> https://www.researchpad.co/article/5ada795d463d7e1f3a3f9aa9

Mycobacterium smegmatis DinB2 is the founder of a clade of Y-family DNA polymerase that is naturally adept at utilizing rNTPs or dNTPs as substrates. Here we investigate the fidelity and lesion bypass capacity of DinB2. We report that DinB2 is an unfaithful DNA and RNA polymerase with a distinctive signature for misincorporation of dNMPs, rNMPs and oxoguanine nucleotides during templated synthesis in vitro. DinB2 has a broader mutagenic spectrum with manganese than magnesium, though low ratios of manganese to magnesium suffice to switch DinB2 to its more mutagenic mode. DinB2 discrimination against incorrect dNTPs in magnesium is primarily at the level of substrate binding affinity, rather than kpol. DinB2 can incorporate any dNMP or rNMP opposite oxo-dG in the template strand with manganese as cofactor, with a kinetic preference for synthesis of an A:oxo-dG Hoogsteen pair. With magnesium, DinB2 is adept at synthesizing A:oxo-dG or C:oxo-dG pairs. DinB2 effectively incorporates deoxyribonucleotides, but not ribonucleotides, opposite an abasic site, with kinetic preference for dATP as the substrate. We speculate that DinB2 might contribute to mycobacterial mutagenesis, oxidative stress and quiescence, and discuss the genetic challenges to linking the polymerase biochemistry to an in vivo phenotype.

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<![CDATA[The key DNA-binding residues in the C-terminal domain of Mycobacterium tuberculosis DNA gyrase A subunit (GyrA)]]> https://www.researchpad.co/article/5b7be21d463d7e7b3349a62c

As only the type II topoisomerase is capable of introducing negative supercoiling, DNA gyrase is involved in crucial cellular processes. Although the other domains of DNA gyrase are better understood, the mechanism of DNA binding by the C-terminal domain of the DNA gyrase A subunit (GyrA-CTD) is less clear. Here, we investigated the DNA-binding sites in the GyrA-CTD of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The results show that Y577, R691 and R745 are among the key DNA-binding residues in M.tuberculosis GyrA-CTD, and that the third blade of the GyrA-CTD is the main DNA-binding region in M.tuberculosis DNA gyrase. The substitutions of Y577A, D669A, R691A, R745A and G729W led to the loss of supercoiling and relaxation activities, although they had a little effect on the drug-dependent DNA cleavage and decatenation activities, and had no effect on the ATPase activity. Taken together, these results showed that the GyrA-CTD is essential to DNA gyrase of M.tuberculosis, and promote the idea that the M.tuberculosis GyrA-CTD is a new potential target for drug design. It is the first time that the DNA-binding sites in GyrA-CTD have been identified.

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<![CDATA[Human replication protein A unfolds telomeric G-quadruplexes]]> https://www.researchpad.co/article/5b7be15f463d7e7b3349a593

G-quadruplex structures inhibit telomerase activity and must be disrupted for telomere elongation during S phase. It has been suggested that the replication protein A (RPA) could unwind and maintain single-stranded DNA in a state amenable to the binding of telomeric components. We show here that under near-physiological in vitro conditions, human RPA is able to bind and unfold G-quadruplex structures formed from a 21mer human telomeric sequence. Analyses by native gel electrophoresis, cross-linking and fluorescence resonance energy transfer indicate the formation of both 1:1 and 2:1 complexes in which G-quadruplexes are unfolded. In addition, quadruplex opening by hRPA is much faster than observed with the complementary DNA, demonstrating that this protein efficiently unfolds G-quartets. A two-step mechanism accounting for the binding of hRPA to G-quadruplexes is proposed. These data point to the involvement of hRPA in regulation of telomere maintenance.

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<![CDATA[RPA and PCNA suppress formation of large deletion errors by yeast DNA polymerase δ]]> https://www.researchpad.co/article/5b7be1af463d7e7b3349a5d1

In fulfilling its biosynthetic roles in nuclear replication and in several types of repair, DNA polymerase δ (pol δ) is assisted by replication protein A (RPA), the single-stranded DNA-binding protein complex, and by the processivity clamp proliferating cell nuclear antigen (PCNA). Here we report the effects of these accessory proteins on the fidelity of DNA synthesis in vitro by yeast pol δ. We show that when RPA and PCNA are included in reactions containing pol δ, rates for single base errors are similar to those generated by pol δ alone, indicating that pol δ itself is by far the prime determinant of fidelity for single base errors. However, the rate of deleting multiple nucleotides between directly repeated sequences is reduced by ∼10-fold in the presence of either RPA or PCNA, and by ≥90-fold when both proteins are present. We suggest that PCNA and RPA suppress large deletion errors by preventing the primer terminus at a repeat from fraying and/or from relocating and annealing to a downstream repeat. Strong suppression of deletions by PCNA and RPA suggests that they may contribute to the high replication fidelity needed to stably maintain eukaryotic genomes that contain abundant repetitive sequences.

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