Examination of the roles of a conserved motif in the PriA helicase in structure-specific DNA unwinding and processivity.
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ABSTRACT:
SUBMITTER: Duckworth AT
PROVIDER: S-EPMC8323898 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA replication restart, the essential process that reinitiates prematurely terminated genome replication reactions, relies on exquisitely specific recognition of abandoned DNA replication-fork structures. The PriA DNA helicase mediates this process in bacteria through mechanisms that remain poorly defined. We report the crystal structure of a PriA/replication-fork complex, which resolves leading-strand duplex DNA bound to the protein. Interaction with PriA unpairs one end of the DNA and sequesters the 3'-most nucleotide from the nascent leading strand into a conserved protein pocket. Cross-linking studies reveal a surface on the winged-helix domain of PriA that binds to parental duplex DNA. Deleting the winged-helix domain alters PriA's structure-specific DNA unwinding properties and impairs its activity in vivo. Our observations lead to a model in which coordinated parental-, leading-, and lagging-strand DNA binding provide PriA with the structural specificity needed to act on abandoned DNA replication forks.
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Project description:MOV10 is an RNA helicase that associates with the RNA-induced silencing complex component Argonaute (AGO), likely resolving RNA secondary structures. MOV10 also binds the Fragile X mental retardation protein to block AGO2 binding at some sites and associates with UPF1, a principal component of the nonsense-mediated RNA decay pathway. MOV10 is widely expressed and has a key role in the cellular response to viral infection and in suppressing retrotransposition. Posttranslational modifications of MOV10 include ubiquitination, which leads to stimulation-dependent degradation, and phosphorylation, which has an unknown function. MOV10 localizes to the nucleus and/or cytoplasm in a cell type-specific and developmental stage-specific manner. Knockout of Mov10 leads to embryonic lethality, underscoring an important role in development where it is required for the completion of gastrulation. MOV10 is expressed throughout the organism; however, most studies have focused on germline cells and neurons. In the testes, the knockdown of Mov10 disrupts proliferation of spermatogonial progenitor cells. In brain, MOV10 is significantly elevated postnatally and binds mRNAs encoding cytoskeleton and neuron projection proteins, suggesting an important role in neuronal architecture. Heterozygous Mov10 mutant mice are hyperactive and anxious and their cultured hippocampal neurons have reduced dendritic arborization. Zygotic knockdown of Mov10 in Xenopus laevis causes abnormal head and eye development and mislocalization of neuronal precursors in the brain. Thus, MOV10 plays a vital role during development, defense against viral infection and in neuronal development and function: its many roles and regulation are only beginning to be unraveled. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
| S-EPMC8799784 | biostudies-literature
Project description:The K-homology (KH) domain is a nucleic acid-binding domain present in many proteins. Recently, we found that the DEAD-box helicase DDX43 contains a KH domain in its N-terminus; however, its function remains unknown. Here, we purified recombinant DDX43 KH domain protein and found that it prefers binding ssDNA and ssRNA. Electrophoretic mobility shift assay and NMR revealed that the KH domain favors pyrimidines over purines. Mutational analysis showed that the GXXG loop in the KH domain is involved in pyrimidine binding. Moreover, we found that an alanine residue adjacent to the GXXG loop is critical for binding. Systematic evolution of ligands by exponential enrichment, chromatin immunoprecipitation-seq, and cross-linking immunoprecipitation-seq showed that the KH domain binds C-/T-rich DNA and U-rich RNA. Bioinformatics analysis suggested that the KH domain prefers to bind promoters. Using 15N-heteronuclear single quantum coherence NMR, the optimal binding sequence was identified as TTGT. Finally, we found that the full-length DDX43 helicase prefers DNA or RNA substrates with TTGT or UUGU single-stranded tails and that the KH domain is critically important for sequence specificity and unwinding processivity. Collectively, our results demonstrated that the KH domain facilitates the substrate specificity and processivity of the DDX43 helicase.
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Project description:The nonstructural NS3 protein of the hepatitis C virus is a multifunctional enzyme with an N-terminal serine protease activity and a C-terminal helicase activity. The helicase is capable of unwinding both DNA and RNA duplexes; however, the overall processivity of the helicase is fairly low. We show here that single-strand binding (SSB) proteins enhance the unwinding processivity of both the NS3 helicase domain (NS3h) and the full-length protease-helicase NS3-4A. The detailed study of the effect of SSB on the DNA unwinding activity of NS3h indicates that the SSB stabilizes the helicase at the unwinding junction and prevents its dissociation. These results suggest a potential role for either cellular or virus-encoded SSB protein in improving the processivity of the NS3 in vivo.
| S-EPMC2249756 | biostudies-literature
Project description:BACKGROUND: Bacterial DNA replication restart pathways facilitate reinitiation of DNA replication following disruptive encounters of a replisome with DNA damage, thereby allowing complete and faithful duplication of the genome. In Neisseria gonorrhoeae, the primosome proteins that catalyze DNA replication restart differ from the well-studied primosome proteins of E. coli with respect to the number of proteins involved and the affinities of their physical interactions: the PriA:PriB interaction is weak in E. coli, but strong in N. gonorrhoeae, and the PriB:DNA interaction is strong in E. coli, but weak in N. gonorrhoeae. In this study, we investigated the functional consequences of this affinity reversal. RESULTS: We report that N. gonorrhoeae PriA's DNA binding and unwinding activities are similar to those of E. coli PriA, and N. gonorrhoeae PriA's helicase activity is stimulated by its cognate PriB, as it is in E. coli. This finding is significant because N. gonorrhoeae PriB's single-stranded DNA binding activity is weak relative to that of E. coli PriB, and in E. coli, PriB's single-stranded DNA binding activity is important for PriB stimulation of PriA helicase. Furthermore, a N. gonorrhoeae PriB variant defective for binding single-stranded DNA can stimulate PriA's helicase activity, suggesting that DNA binding by PriB might not be important for PriB stimulation of PriA helicase in N. gonorrhoeae. We also demonstrate that N. gonorrhoeae PriB stimulates ATP hydrolysis catalyzed by its cognate PriA. This activity of PriB has not been observed in E. coli, and could be important for PriB stimulation of PriA helicase in N. gonorrhoeae. CONCLUSIONS: The results of this study demonstrate that a bacterial PriB homolog with weak single-stranded DNA binding activity can stimulate the DNA unwinding activity of its cognate PriA helicase. While it remains unclear if N. gonorrhoeae PriB's weak DNA binding activity is required for PriB stimulation of PriA helicase, the ability of PriB to stimulate PriA-catalyzed ATP hydrolysis could play an important role. Thus, the weak interaction between N. gonorrhoeae PriB and DNA might be compensated for by the strong interaction between PriB and PriA, which could result in allosteric activation of PriA's ATPase activity.
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Project description:Phage T7 helicase unwinds double-stranded DNA (dsDNA) by encircling one strand while excluding the complementary strand from its central channel. When T7 helicase translocates on single-stranded DNA (ssDNA), it has kilobase processivity; yet, it is unable to processively unwind linear dsDNA, even 60 base-pairs long. Particularly, the GC-rich dsDNAs are unwound with lower amplitudes under single-turnover conditions. Here, we provide evidence that T7 helicase switches from ssDNA to dsDNA during DNA unwinding. The switching propensity is higher when dsDNA is GC-rich or when the 3'-overhang of forked DNA is <15 bases. Once helicase encircles dsDNA, it travels along dsDNA and dissociates from the end of linear DNA without strand separation, which explains the low unwinding amplitude of these substrates. Trapping the displaced strand with ssDNA binding protein or changing its composition to morpholino oligomer that does not interact with helicase increases the unwinding amplitude. We conclude that the displaced strand must be continuously excluded and kept away from the central channel for processive DNA unwinding. The finding that T7 helicase can switch from ssDNA to dsDNA binding mode during unwinding provides new insights into ways of limiting DNA unwinding and triggering fork regression when stalled forks need to be restarted.
| S-EPMC3627605 | biostudies-literature
Project description:Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of the conserved lysine in the Walker A motif abolished helicase activity, implying that not the N-terminal portion, but the helicase domain is solely responsible for the enzyme's unwinding activity. In addition, we demonstrate a novel stimulation of RecQ4's helicase activity by replication protein A, similar to that of RecQ1, BLM, WRN, and RecQ5. Together, these data indicate that specific biochemical activities and protein partners of RecQ4 are conserved with those of the other RecQ helicases.
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Project description:Recent studies suggest a link between infection by Zika virus (ZIKV) and the development of neurological complications. The lack of ZIKV-specific therapeutics has alarmed healthcare professionals worldwide. Here, crystal structures of apo and AMPPNP- and Mn2+-bound forms of the essential helicase of ZIKV refined to 1.78 and 1.3?Å resolution, respectively, are reported. The structures reveal a conserved trimodular topology of the helicase. ATP and Mn2+ are tethered between two RecA-like domains by conserved hydrogen-bonding interactions. The binding of ligands induces the movement of backbone C? and side-chain atoms. Numerous solvent molecules are observed in the vicinity of the AMPPNP, suggesting a role in catalysis. These high-resolution structures could be useful for the design of inhibitors targeting the helicase of ZIKV for the treatment of infections caused by ZIKV.
| S-EPMC5894106 | biostudies-literature
Project description:Primosomal protein A (PriA) is a member of helicase SuperFamily 2. Its role in vivo is to reload the primosome onto resurrected replication forks resulting in the restart of the previously stalled DNA replication process. Single-stranded DNA-binding protein (SSB) plays a key role in mediating activities at replication forks and interacts both physically and functionally with PriA. To gain a mechanistic insight into the PriA-SSB interaction, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of PriA in vitro in the presence of fork substrates. The results demonstrate that SSB enhances the ability of PriA to discriminate between fork substrates as much as 140-fold. This is due to a significant increase in the catalytic efficiency of the helicase induced by SSB. This interaction is species-specific as bacteriophage gene 32 protein cannot substitute for the Escherichia coli protein. SSB, while enhancing the activity of PriA on its preferred fork decreases both the affinity of the helicase for other forks and the catalytic efficiency. Central to the stimulation afforded by SSB is the unique ability of PriA to bind with high affinity to the 3'-OH placed at the end of the nascent leading strand at the fork. When both the 3'-OH and SSB are present, the maximum effect on the ATPase activity of the helicase is observed. This ensures that PriA will load onto the correct fork, in the right orientation, thereby ensuring that replication restart is directed to only the template lagging strand.
| S-EPMC8246471 | biostudies-literature
Project description:Licensing of eukaryotic origins of replication requires DNA loading of two copies of the Mcm2-7 replicative helicase to form a head-to-head double-hexamer, ensuring activated helicases depart the origin bidirectionally. To understand the formation and importance of this double-hexamer, we identified mutations in a conserved and essential Mcm4 motif that permit loading of two Mcm2-7 complexes but are defective for double-hexamer formation. Single-molecule studies show mutant Mcm2-7 forms initial hexamer-hexamer interactions; however, the resulting complex is unstable. Kinetic analyses of wild-type and mutant Mcm2-7 reveal a limited time window for double-hexamer formation following second Mcm2-7 association, suggesting that this process is facilitated. Double-hexamer formation is required for extensive origin DNA unwinding but not initial DNA melting or recruitment of helicase-activation proteins (Cdc45, GINS, Mcm10). Our findings elucidate dynamic mechanisms of origin licensing, and identify the transition between initial DNA melting and extensive unwinding as the first initiation event requiring double-hexamer formation.
| S-EPMC6701924 | biostudies-literature