Frequent exchange of the DNA polymerase during bacterial chromosome replication
Abstract
The replisome is a multiprotein machine that carries out DNA replication. In Escherichia coli, a single pair of replisomes is responsible for duplicating the entire 4.6 Mbp circular chromosome (Beattie and Reyes-Lamothe, 2015). In vitro studies of reconstituted E. coli replisomes have attributed this remarkable processivity to the high stability of the replisome once assembled on DNA (Tanner et al., 2011, Yao et al., 2009, Kim et al., 1996b). By examining replisomes in live E. coli with fluorescence microscopy, we found that the Pol III* subassembly frequently disengages from the replisome during DNA synthesis and exchanges with free copies from solution. In contrast, the DnaB helicase associates stably with the replication fork, providing the molecular basis for how the E. coli replisome can maintain high processivity and yet possess the flexibility to bypass obstructions in template DNA. Our data challenges the widely-accepted semi-discontinuous model of chromosomal replication, instead supporting a fully discontinuous mechanism in which synthesis of both leading and lagging strands is frequently interrupted.
Article and author information
Author details
Funding
Natural Sciences and Engineering Research Council of Canada (Discovery Grant,435521-2013)
- Thomas R Beattie
- Nitin Kapadia
- Rodrigo Reyes-Lamothe
Canada Research Chairs (Tier II,950-228994)
- Rodrigo Reyes-Lamothe
Canadian Institutes of Health Research (Operating Grant,142473)
- Thomas R Beattie
- Nitin Kapadia
- Rodrigo Reyes-Lamothe
Canada Foundation for Innovation (Leaders Oportunity Fund,228994)
- Thomas R Beattie
- Nitin Kapadia
- Rodrigo Reyes-Lamothe
Wellcome (Junior Research Fellowship)
- Stephan Uphoff
Biotechnology and Biological Sciences Research Council (BBSRC# BB/N006453/1)
- Adam JM Wollman
- Mark C Leake
Medical Research Council (MRC# MR/K01580X/1)
- Adam JM Wollman
- Mark C Leake
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- James M Berger, Johns Hopkins University School of Medicine, United States
Version history
- Received: September 22, 2016
- Accepted: March 28, 2017
- Accepted Manuscript published: March 31, 2017 (version 1)
- Version of Record published: April 24, 2017 (version 2)
Copyright
© 2017, Beattie et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 4,304
- views
-
- 829
- downloads
-
- 99
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.