Investigations of π Initiator Protein-mediated Interaction between Replication Origins α and γ of the Plasmid R6K

Abstract

A typical plasmid replicon of Escherichia coli, such as ori ␥ of R6K, contains tandem iterons (iterated initiator protein binding sites), an AT-rich region that melts upon initiator-iteron interaction, two binding sites for the bacterial initiator protein DnaA, and a binding site for the DNA-bending protein IHF. R6K also contains two structurally atypical origins called ␣ and ␤ that are located on either side of ␥ and contain a single and a half-iteron, respectively. Individually, these sites do not bind to initiator protein but access it by DNA looping-mediated interaction with the seven -bound ␥ iterons. The protein exists in 2 interconvertible forms: inert dimers and active monomers. Initiator dimers generally function as negative regulators of replication by promoting iteron pairing ("handcuffing") between pairs of replicons that turn off both origins. Contrary to this existing paradigm, here we show that both the dimeric and the monomeric are necessary for ori ␣-driven plasmid maintenance. Furthermore, efficient looping interaction between ␣ and ␥ or between 2 ␥ iterons in vitro also required both forms of . Why does ␣-␥ iteron pairing promote ␣ activation rather than repression? We show that a weak, transitory ␣-␥ interaction at the iteron pairs was essential for ␣-driven plasmid maintenance. Swapping the ␣ iteron with one of ␥ without changing the original sequence context that caused enhanced looping in vitro caused a significant inhibition of ␣-mediated plasmid maintenance. Therefore, the affinity of ␣ iteron for -bound ␥ and not the sequence context determined whether the origin was activated or repressed.

Plasmid R6K replication control

“…π exhibits remarkable structural and functional plasticity in its interactions with diverse DNA target sites, and these different interactions are the basis for its different biological functions: replication initiator, replication inhibitor, transcription (auto)-repressor and origin selection (α, β or γ) factor (Germino and Bastia, 1983b; Germino and Bastia, 1983a; Urh et al, 1998; Krüger et al, 2004b; Saxena et al, 2010a; Saxena et al, 2010b). Encoded by R6K's pir gene, π is a member of the Rep family of replication initiator proteins with sequence similarities that extend even to eukaryotic proteins (Giraldo and Díaz-Orejas, 2001).…”

“…The data and conclusions presented by Kunnimalaiyaan et al (2004; 2007) and Swan et al (2006) are in good agreement but with some variation. For example, research by the former group did not investigate all of the amino acids implicated in DNA contact by the crystal structure, two of which (Arg75 and Asp226) received additional support in later mutation analyses (Saxena et al, 2010b). In contrast, the work of Swan et al assigns no role in iteron binding to Gly211, which would seem to have a critical functional role based on Rep protein sequence conservation.…”

“…Unlike most Rep/iteron plasmids, an important characteristic of R6K (a.k.a., RSF1040) is that it has, not 1, but 3 functional origins of replication and, typically, the ori s do not fire concurrently (Crosa, 1980). Research designed to elucidate the mechanisms that regulate ori selection has recently been undertaken (Saxena et al, 2010a; Saxena et al, 2010b) and more work in this area will be necessary for a full appreciation of the biology of this antibiotic resistance plasmid. It will also contribute to our understanding of replication in a broader context, as multiple replication origins are typical of eukaryotic replicons and are also seen outside of Eukarya (e.g., Huberman and Riggs, 1968; Blumenthal et al, 1974; Newlon et al, 1974; Bourguignon et al, 1976; Robinson et al, 2004).…”

“…Although the γ ori of R6K has been the most studied, its other origins, α and β are preferentially utilized in vivo (Crosa et al, 1976; Crosa, 1980). Curiously, however, the γ ori core is necessary for the activation of the other two ori s. π protein facilitates interaction between the iterons in γ ori and iteron-like sequences in ori s α and β (Mukherjee et al, 1988b; Miron et al, 1992; Saxena et al, 2010a; Saxena et al., 2010b). This results in a looping of the plasmid DNA, which is believed to transmit the replication signal from the internal iteron cluster (7 DRs) to the outlying R6K ori s (Mukherjee et al, 1988a; Mukherjee et al, 1988b).…”

“…This results in a looping of the plasmid DNA, which is believed to transmit the replication signal from the internal iteron cluster (7 DRs) to the outlying R6K ori s (Mukherjee et al, 1988a; Mukherjee et al, 1988b). Studies of the interaction between γ and α ori s suggest that both monomers and dimers of π protein participate in the looping process (Saxena et al, 2010a; Saxena et al, 2010b). Thus, related yet distinct protein-protein and protein-DNA interactions appear to be crucial to the function of each replication origin.…”