Clever DNA tricks | Ars Technica doi:10.1016/j.molcel.2020.08.014, Kose, H. B., Xie, S., Cameron, G., Strycharska, M. S., and Yardimci, H. (2020). McGlynn, P., Savery, N. J. A nanotensioner is a short DNA duplex designed to apply force to both ends of the unwinding template overhangs (Figure 5C). doi:10.1093/nar/gkaa320, Duderstadt, K. E., Geertsema, H. J., Stratmann, S. A., Punter, C. M., Kulczyk, A. W., Richardson, C. C., et al. Pomerantz, R. T. & ODonnell, M. What happens when replication and transcription complexes collide? It is possible that all these replicative helicases are passive, and thus far we have not been able to identify the proper model. 116, 684693. 1b). Focusing such methods on replicative helicases has provided a unique window to observe intra- and inter-molecular variation in helicase activity within the context of the replisome. B.S. From these experiments it is possible to investigate the coordination of hexameric subunits by quantifying and comparing intra- and inter-molecular dynamics during unwinding. In the unzipping force analysis of helicase and DNAP association, after the detection of helicase loading and unwinding, the remaining dsDNA was mechanically unzipped at an extremely fast velocity of 2000bp/s to probe the potential interactions at the fork. DNA helicases are enzymes that coordinate nucleotide hydrolysis with their translocation and unwinding of duplex nucleic acids. Arm 2 (2013 bp) was PCR amplified from plasmid pBR322 (NEB, Ipswich, MA) using a biotin-labeled primer. 6 Articles, This article is part of the Research Topic, The Single-Molecule Toolbox to Examine Helicases, Dynamics of Helicase-Replisome Interactions, https://doi.org/10.3389/fmolb.2021.741718. Structural studies have arguably made the greatest contribution to our knowledge of helicase unwinding mechanisms. Thus, it is important to determine the factors that contribute to the stepping action and step size of a helicase. A prerequisite for a replisome to resume replication after the lesion is the acquisition of a primer, which allows DNA polymerase (DNAP) to re-initiate DNA synthesis. Surprisingly, we found that in the presence of the non-replicating DNAP, T7 helicase unwound DNA processively without detectable slippage through the entire dsDNA available (~2500bp) (Fig. 279, 2572125728. Google Scholar. 84, 13081316 (2003). Unwinding rates were determined between slippage events (if any). Romano, L. J., Tamanoi, F. & Richardson, C. C. Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins: requirement for T7 RNA polymerase. Likewise, single-molecule analysis of replisomal exchange identified that replicative helicases exchange very infrequently and instead are stably maintained throughout replication. Experiments were carried out in the presence of helicase, DNAP, and 0.5mM dNTP (each) under 5pN of force. (E) Single-molecule fluorescence recovery after photobleaching (FRAP) method can be used to quantify protein exchange in the form of recovered fluorescence following a deliberate bleaching event. Natl. Microbiol. DNA Synthesis Provides the Driving Force to Accelerate DNA Unwinding by a Helicase. Sun, B. In all replisomes, the primary responsibility of the replicative helicase is to unwind double-stranded parental DNA into two template strands for copying. Commun. In this sense, the replicative helicase becomes the anchor of the replisome, ensuring processive replication and providing a constant binding site for other exchanging proteins (Figures 7A,B). Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication. 267, 1501315021 (1992). Only unwinding activity is examined in this assay and unlike the T7 gp4, the T4 helicase and primase are separate entities that can dissociate if need be. The Replication Time of the Escherichia coli K12 Chromosome as a Function of Cell Doubling Time. Biochemistry 36, 82318242 (1997). 1a). 8). The following sub-sections compile our current understanding of unwinding mechanisms and highlight the progress derived from single-molecule studies. Its position in nm showed in Fig. The FRET efficiency indicates the hexameric ring state, where high FRET denotes a closed ring and low FRET denotes an open ring. You are using a browser version with limited support for CSS. For example, methods such as single-molecule nanopores or hybrid fluorescence and force measurements have been effective to examine other motor enzymes (reviewed in Mohapatra et al., 2020). Helicase Stepping Investigated with One-Nucleotide Resolution Fluorescence Resonance Energy Transfer. Alternatively, some of these helicases could be partially active, but we do not currently possess techniques with the necessary discriminatory power to confirm this hypothesis. Gp41, Gp4, DnaB, and CMG all exhibit this propertyin distinct contrast to that of a known active helicase like RecQ (Manosas et al., 2010). In the future, we expect to see further advancements in the field of single-molecule replicative helicase research as discussed here. Perspect. Chem. (D) Typical FRET unwinding traces for the gp4 helicase, where a nanotensioner is incorporated into the DNA template. find in their gp4 structures that the NTPase active site is tightest at the last subunit position in the spiral. Article Instances of low FRET (black lines) correlate with MCM27 binding, indicating that the ring briefly opens as it encircles DNA during loading (Ticau et al., 2017). Single-molecule experiments on helicase and non-replicating DNAP collision with a TEC. T7 RNAP was overexpressed in E. coli strain BL21/pAR1219 and purified using three chromatographic columns consisting of SP-Sephadex, CM-Sephadex, and DEAD- Sephacel57, 58. J. Biol. PubMed (2012). Quart. DNA Replication: Steps, Process, Diagram and Simple Explanation Structure of Eukaryotic CMG Helicase at a Replication fork and Implications to Replisome Architecture and Origin Initiation. Biol. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. Upon collision, several types of behavior were observed. Experiments were conducted in a climate-controlled room at a temperature of 23.3C, but owing to local laser trap heating the temperature increased slightly to 251C63. In a recent structure, the T7 gp4 helicase and gp5 polymerase are positioned perpendicular to each other at the fork, further suggesting there is some coupling effect during unwinding (Gao et al., 2019); however, mutational evidence suggests that such synergy is not dependent on the physical connection between the helicase and polymerase (Stano et al., 2005). The force-rise is thus attributed to the helicase and DNAP interactions across the fork junction. Ma et al. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Patel, S. S., Rosenberg, A. H., Studier, F. W. & Johnson, K. A. Large scale purification and biochemical characterization of T7 primase/helicase proteins. Proposed T7 replication re-initiation model. Phys. Natl. 11, 688. doi:10.1038/s41467-020-14577-6, Zhang, Z., Spiering, M. M., Trakselis, M. A., Ishmael, F. T., Xi, J., Benkovic, S. J., et al. Symp. We should also question the robustness of the passive unwinding model. The helicase unwinds, loses grip, slips, re-grips, and resumes unwinding. The model is extremely sensitive to the value of step size and slippage frequencyparameters that are both very difficult to measure. Biol. This nucleotide condition supports DNA unwinding but does not support DNA replication because dNTPs are missing. These observations exhibit T7 helicases novel role in replication re-initiation. Biophys. Methods Enzymol. RecQ is a recombinationspecific DNA helicase from the SF2 family. Mol. Nat. Also, the eukaryotic helicase and primase should be examined. For experiments described in Fig. ), Shanghai Pujiang Program (16PJ1406900 to B.S. Mutations in enzymes with helicase activity result in a variety of human genetic diseases. An RNA primer is synthesized, and is elongated by the DNA polymerase. In archaeal cells, the same mutation results in . Natl. 1), we favor the possibility that DNAP was present at the fork, but the DNAPhelicase or DNAPDNA interactions were transiently lost at the moment of detection. The replicative helicases of the model systems bacteriophages T4 . Their work was able to identify the order of key events during T4 initiation. 11, 683687 (2010). 102, 32543259. Same experimental conditions were used as in c. For clarity, traces have been shifted along the time axis. The chemical directionality of DNA poses a unique challenge when it comes to replicating the chromosomes of an organism. Cell 151, 267277. 1), it is unclear whether the non-replicating helicaseDNAP complex is capable of overcoming the TEC barrier. The DNA template for helicase unwinding and unzipping consisted of a 1.1 kbp anchoring segment and a 4.1 kbp unwinding segment (Supplementary Fig. The non-replicating DNAP stimulates the unwinding activity of the helicase by direct interactions. 22, 948952. The Structure of a DnaB-Family Replicative Helicase and its Interactions with Primase. Thank you for visiting nature.com. doi:10.1038/nature04317, Leipe, D. D., Aravind, L., and Koonin, E. V. (1999). Helicase and Polymerase Move Together Close to the fork junction and Copy DNA in One-Nucleotide Steps. Similar to T7 gp4, the DnaB translocation state is also observed as a spiral staircase with each subunit holding a NTP at the subunit interface and in contact with 2nt of the A-form-like DNA backbone (Figure 2B) (Itsathitphaisarn et al., 2012). Interestingly, further analysis of the single-molecule kinetics showed that the dwell time between steps was best described by a gamma distribution rather than an exponential one. doi:10.1093/nar/gkq273, Matson, S. W., and Richardson, C. C. (1983). The bacteriophage T7 replisome is a simple model system which provides a powerful in vitro system to decipher detailed mechanisms of DNA replication19,20,21,22,23. Such a fit suggests that there are several rate-limiting kinetic steps hidden within bursts of 23 bp of unwinding. Nature 462, 854855. It is proposed that this dam-and-diversion process may explain how steric exclusion occurs for CMG. drafted the manuscript. doi:10.1074/jbc.272.29.18425, ODonnell, M. E., and Li, H. (2018). Structure of the Eukaryotic Replicative CMG Helicase Suggests a Pumpjack Motion for Translocation. (2018). On the fork substrate containing a stalled TEC, we observed run-off products in the presence of dNTPs only when both helicase and DNAP were present. Single-molecule Analysis and Engineering of DNA Motors. It is expected to either act behind the replisome or interact with CMG indirectly via the primase Pol , or the Ctf4 organizing protein. PubMed Brennan, L. D., Forties, R. A., Patel, S. S. & Wang, M. D. DNA looping mediates nucleosome transfer. Structures and Operating Principles of the Replisome. 111, 25622569. A Conserved Mcm4 Motif Is Required for Mcm2-7 Double-Hexamer Formation and Origin DNA Unwinding. In fact, many lines of evidence in vitro and in vivo support the occurrence of both co-directional and head-on collisions5,6,7,8. Further to this, we had already discussed the function and structure of DNA primase that provides RNA primer for starting DNA replication. Mol. In these species, the replicative helicases, gp4 in T7 and gp41 in T4, are superfamily (SF) four helicases, which are characterized as homo-hexameric rings that employ a RecA-like motor domain to power translocation along single-stranded (ss) DNA in the 53 direction and concomitantly unwind duplex DNA by exclusion of the other strand. For a detailed review of E. coli DNA replication, see Lewis et al. Sun, B. et al. TABLE 1. On templates containing no GC base pairs, G40P unwinding exhibits a rapid decrease in FRET. doi:10.1093/nar/gks254, Manosas, M., Spiering, M. M., Zhuang, Z., Benkovic, S. J., and Croquette, V. (2009). Also of note are the multi-color colocalization methods that, although conceptually simple, are highly effective at reporting on the interaction network of replicative helicases even in complex reactions (Figure 1D). During DNA replication, the enzyme helicase unwinds the DNA double helix by disrupting the hydrogen bonds that keep it together. We conclude that helicase in association with a non-replicating DNAP forms a strong motor complex at the fork, capable of displacing an RNAP. Acad. USA 99, 1353813543 (2002). 120, 3678. Helicase promotes replication re-initiation from an RNA transcript | Nature Communications Article Published: 13 June 2018 Helicase promotes replication re-initiation from an RNA transcript Bo. Nat. Nevin, P., Gabbai, C. C. & Marians, K. J. Replisome-mediated translesion synthesis by a cellular replicase. Mol. It is called a fork because the structure resembles a two-pronged fork. Rezipping is independent of force, but equivalent to the ssDNA translocation rate (
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helicase in dna replication