The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol α-primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol α-primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.
Ribonucleotide incorporation characteristics around yeast autonomously replicating sequences reveal the labor division of replicative DNA polymerases
Ribonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARS’s) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARS’s, highlighting the Pol δ - Pol ε handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol δ and Pol ε.
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- Award ID(s):
- 1764406
- NSF-PAR ID:
- 10253601
- Date Published:
- Journal Name:
- bioRxiv
- ISSN:
- 2692-8205
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The unequal partitioning of molecules and organelles during cell division results in daughter cells with different fates. An extreme example is female meiosis, in which consecutive asymmetric cell divisions give rise to 1 large oocyte and 2 small polar bodies with DNA and minimal cytoplasm. Here, we test the hypothesis that during an asymmetric cell division during spermatogenesis of the nematode Auanema rhodensis, the late segregating X chromatids orient the asymmetric partitioning of cytoplasmic components. In previous studies, the secondary spermatocytes of wild-type XO males were found to divide asymmetrically to generate functional spermatids that inherit components necessary for sperm viability and DNA-containing residual bodies that inherit components to be discarded. Here we extend that analysis to 2 novel contexts. First, the isolation and analysis of a strain of mutant XX pseudomales revealed that such animals have highly variable patterns of X-chromatid segregation. The pattern of late segregating X chromatids nevertheless predicted the orientation of organelle partitioning. Second, while wild-type XX hermaphrodites were known to produce both 1X and 2X sperm, here, we show that spermatocytes within specific spermatogonial clusters exhibit 2 different patterns of X-chromatid segregation that correlate with distinct patterns of organelle partitioning. Together this analysis suggests that A. rhodensis has coopted lagging X chromosomes during anaphase II as a mechanism for determining the orientation of organelle partitioning.
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DNA polymerase β (pol β ) is a member of the X- family of DNA polymerases that catalyze the distributive addition of nucleoside triphosphates during base excision DNA repair. Previous studies showed that the enzyme was phosphorylated in vitro with PKC at two serines (44 and 55), causing loss of DNA polymerase activity but not DNA binding. In this work, we have investigated the phosphorylation-induced conformational changes in DNA polymerase β in the presence of Mg ions. We report a comprehensive atomic resolution study of wild type and phosphorylated DNA polymerase using molecular dynamics (MD) simulations. The results are examined via novel methods of internal dynamics and energetics analysis to reveal the underlying mechanism of conformational transitions observed in DNA pol β . The results show drastic conformational changes in the structure of DNA polymerase β due to S44 phosphorylation. Phosphorylation-induced conformational changes transform the enzyme from a closed to an open structure. The dynamic cross-correlation shows that phosphorylation enhances the correlated motions between the different domains. Centrality network analysis reveals that the S44 phosphorylation causes structural rearrangements and modulates the information pathway between the Lyase domain and base pair binding domain. Further analysis of our simulations reveals that a critical hydrogen bond (between S44 and E335) disruption and the formation of three additional salt bridges are potential drivers of these conformational changes. In addition, we found that two of these additional salt bridges form in the presence of Mg ions on the active sites of the enzyme. These results agree with our previous study of DNA pol β S44 phosphorylation without Mg ions which predicted the deactivation of DNA pol β . However, the phase space of structural transitions induced by S44 phosphorylation is much richer in the presence of Mg ions.more » « less
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Daròs, José-Antonio (Ed.)Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription.more » « less
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Simon, Anne E. (Ed.)ABSTRACT Long noncoding RNAs (lncRNAs) of virus origin accumulate in cells infected by many positive-strand (+) RNA viruses to bolster viral infectivity. Their biogenesis mostly utilizes exoribonucleases of host cells that degrade viral genomic or subgenomic RNAs in the 5′-to-3′ direction until being stalled by well-defined RNA structures. Here, we report a viral lncRNA that is produced by a novel replication-dependent mechanism. This lncRNA corresponds to the last 283 nucleotides of the turnip crinkle virus (TCV) genome and hence is designated tiny TCV subgenomic RNA (ttsgR). ttsgR accumulated to high levels in TCV-infected Nicotiana benthamiana cells when the TCV-encoded RNA-dependent RNA polymerase (RdRp), also known as p88, was overexpressed. Both (+) and (−) strand forms of ttsgR were produced in a manner dependent on the RdRp functionality. Strikingly, templates as short as ttsgR itself were sufficient to program ttsgR amplification, as long as the TCV-encoded replication proteins p28 and p88 were provided in trans . Consistent with its replicational origin, ttsgR accumulation required a 5′ terminal carmovirus consensus sequence (CCS), a sequence motif shared by genomic and subgenomic RNAs of many viruses phylogenetically related to TCV. More importantly, introducing a new CCS motif elsewhere in the TCV genome was alone sufficient to cause the emergence of another lncRNA. Finally, abolishing ttsgR by mutating its 5′ CCS gave rise to a TCV mutant that failed to compete with wild-type TCV in Arabidopsis . Collectively, our results unveil a replication-dependent mechanism for the biogenesis of viral lncRNAs, thus suggesting that multiple mechanisms, individually or in combination, may be responsible for viral lncRNA production. IMPORTANCE Many positive-strand (+) RNA viruses produce long noncoding RNAs (lncRNAs) during the process of cellular infections and mobilize these lncRNAs to counteract antiviral defenses, as well as coordinate the translation of viral proteins. Most viral lncRNAs arise from 5′-to-3′ degradation of longer viral RNAs being stalled at stable secondary structures. Here, we report a viral lncRNA that is produced by the replication machinery of turnip crinkle virus (TCV). This lncRNA, designated ttsgR, shares the terminal characteristics with TCV genomic and subgenomic RNAs and overaccumulates in the presence of moderately overexpressed TCV RNA-dependent RNA polymerase (RdRp). Furthermore, templates that are of similar sizes as ttsgR are readily replicated by TCV replication proteins (p28 and RdRp) provided from nonviral sources. In summary, this study establishes an approach for uncovering low abundance viral lncRNAs, and characterizes a replicating TCV lncRNA. Similar investigations on human-pathogenic (+) RNA viruses could yield novel therapeutic targets.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1101/2020.08.27.270728
