Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studied Escherichia coli replication origin, oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5TGTGNAT͞AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, I2 and I3, with sequence 5TG͞TGGATCAG͞A. We now show that sites I2 and I3 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and open oriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation. O ne of the earliest steps in triggering new rounds of DNA synthesis requires binding of initiator proteins to replication origin DNA (1). Several origin-binding proteins in eukaryotes, archea, and eubacteria are members of the AAAϩ family of ATPases, which are active in the ATP-bound form and are inactivated by hydrolysis of ATP to ADP (1-4). These proteins form complexes that mediate subsequent steps of initiation, including DNA strand separation and recruitment of replisome components (1). The recent discovery of structural conservation in AAAϩ initiation proteins in bacteria, archea, and yeast (3-5) suggests that aspects of the initiation mechanism may be similar across all domains of life. However, additional information on the specific function of ATP in the early steps of initiation is required before common initiation mechanisms can be inferred.For a variety of bacterial chromosomal and plasmid origins, as well as some eukaryotic viral origins, the first step of initiation requires binding of multiple copies of an initiator protein that serves to melt the DNA duplex in an AT-rich region (1, 6). This process has been extensively studied in Escherichia coli, where initial strand opening requires multiple copies of the AAAϩ initiator DnaA (7,8). Only ATP-DnaA is active in formation of the open complex (8, 9). Within oriC are five 9-mer DnaAbinding sites (R boxes; see Fig. 1A) with consensus sequence 5ЈTGTGNAT͞AAA (10, 11). Although R boxes have different affinities for DnaA based on slight differences in the nucleotide sequence, each R box binds inactive DnaA-ADP and active DnaA-ATP with equal affinity (8, 12). Based on this finding, two scenarios seem likely with regard to the requirement for DnaA-ATP in oriC unwinding. One possibility is that DnaA-ATP and Dna...
SummaryPrior to initiating DNA synthesis, Escherichia coli oriC switches from ORC, comprising initiator DnaA bound at three high-affinity sites, to pre-RC, when additional DnaA molecules interact with low-affinity sites. Two types of low-affinity sites exist: R boxes that bind DnaA-ATP and DnaA-ADP with equal affinity, and I-sites with a three-to fourfold preference for DnaA-ATP. To assess the regulatory role of weak DnaA interactions during pre-RC assembly in vivo, we compared the behaviour of plasmid-borne wild-type oriC with mutants having an increased or decreased number of DnaA-ATP discriminatory I-sites. Increasing the number of discriminatory sites by replacing R5M with I2 inactivated extrachromosomal oriC function. Mutants with no discriminatory sites perturbed host growth and rapidly replaced wild-type chromosomal oriC, but normal function returned if one I-site was restored at either the I2, I3 or R5M position. These observations are consistent with assembly of E. coli pre-RC in vivo from mixtures of DnaA-ATP and DnaA-ADP, with I-site interactions coupling pre-RC assembly to DnaA-ATP levels.
The nucleoprotein complex formed on oriC, the Escherichia coli replication origin, is dynamic. During the cell cycle, high levels of the initiator DnaA and a bending protein, IHF, bind to oriC at the time of initiation of DNA replication, while binding of Fis, another bending protein, is reduced. In order to probe the structure of nucleoprotein complexes at oriC in more detail, we have developed an in situ footprinting method, termed drunken-cell footprinting, that allows enzymatic DNA modifying reagents access to intracellular nucleoprotein complexes in E.coli, after a brief exposure to ethanol. With this method, we observed in situ binding of Fis to oriC in exponentially growing cells, and binding of IHF to oriC in stationary cells, using DNase I and Bst NI endonuclease, respectively. Increased binding of DnaA to oriC in stationary phase was also noted. Because binding of DnaA and IHF results in unwinding of oriC in vitro, P1 endonuclease was used to probe for intracellular unwinding of oriC. P1 cleavage sites, localized within the 13mer unwinding region of oriC ', were dramatically enhanced in stationary phase on wild-type origins, but not on mutant versions of oriC unable to unwind. These observations suggest that most oriC copies become unwound during stationary phase, forming an initiation-like nucleoprotein complex.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.