The Different Faces of Rolling-Circle Replication and Its Multifunctional Initiator Proteins

Abstract

Horizontal gene transfer (HGT) contributes greatly to the plasticity and evolution of prokaryotic and eukaryotic genomes. The main carriers of foreign DNA in HGT are mobile genetic elements (MGEs) that have extremely diverse genetic structures and properties. Various strategies are used for the maintenance and spread of MGEs, including (i) vegetative replication, (ii) transposition (and other types of recombination), and (iii) conjugal transfer. In many MGEs, all of these processes are dependent on rolling-circle replication (RCR). RCR is one of the most well characterized models of DNA replication. Although many studies have focused on describing its mechanism, the role of replication initiator proteins has only recently been subject to in-depth analysis, which indicates their involvement in multiple biological process associated with RCR. In this review, we present a general overview of RCR and its impact in HGT. We focus on the molecular characteristics of RCR initiator proteins belonging to the HUH and Rep_trans protein families. Despite analogous mechanisms of action these are distinct groups of proteins with different catalytic domain structures. This is the first review describing the multifunctional character of various types of RCR initiator proteins, including the latest discoveries in the field. Recent reports provide evidence that (i) proteins initiating vegetative replication (Rep) or mobilization for conjugal transfer (Mob) may also have integrase (Int) activity, (ii) some Mob proteins are capable of initiating vegetative replication (Rep activity), and (iii) some Rep proteins can act like Mob proteins to mobilize plasmid DNA for conjugal transfer. These findings have significant consequences for our understanding of the role of RCR, not only in DNA metabolism but also in the biology of many MGEs.

Keywords: conjugal transfer; horizontal gene transfer; mobile genetic elements; multifunctional protein; rolling-circle replication; transposition.

FIGURE 1

Mechanism of rolling-circle replication. (A) A model for RCR based on studies of plasmid pT181. Separated cycles of leading (+) and lagging (–) DNA strand synthesis (initiated at the dso and sso origins, respectively) are marked by red dotted arrows. The individual steps in the synthesis are described in the Figure and details are given in the text. The RCR initiation protein (RepC) initiates (+) strand replication at the nick site (marked by a yellow rectangle). Dashed blue lines indicate the nascent DNA strand and blue arrows show the direction of synthesis. Black dotted arrows indicate nucleophilic attack of the catalytic tyrosine residue on the phosphodiester bond in the DNA or of the free 3′-OH DNA end on the phosphotyrosine bond between the enzyme and the DNA. (B) ssDNA breakage and joining reactions catalyzed by HUH and Rep_trans endonucleases at the dso. The nick site exposed in a hairpin-like structure is shown by a yellow rectangle. The red star indicates the phosphotyrosine bond between the enzyme and the 5′-end of the DNA (see text for details). The reaction catalyzed by HUH/Rep_trans is reversible since the released 3′-OH DNA end could acts as a nucleophile on the phosphotyrosine bond (adopted from Chandler et al., 2013; Pastrana et al., 2016).

FIGURE 2

Rolling-circle replication (RCR) in conjugal transfer. The individual stages of DNA synthesis during plasmid conjugal transfer (1–5). The RCR initiation protein (relaxase) containing two conserved tyrosine residues, Y1 and Y2, initiates (+) strand replication at the nick site (marked by a yellow rectangle). Both Y1 and Y2 may be involved in DNA processing during the transfer (plasmid R388) (3a, 4a), or only one of them – Y1 (plasmid F) (3b, 4b) (see text for details). Dashed blue lines indicate the nascent DNA strand and blue arrows show the direction of synthesis. Black dotted arrows indicate nucleophilic attack of the catalytic tyrosine residue on the phosphodiester bond in the DNA or of the free 3′-OH DNA end on the phosphotyrosine bond between the enzyme and the DNA (adopted from Chandler et al., 2013).

FIGURE 3

Different applications of RCR. Models for rolling-hairpin replication (RHR) of adeno-associated virus (AAV) and rolling-circle transposition (RCT) of IS91/HeliBat and Helitrons. The individual stages of replication are described in the Figure and details are given in the text. ITRs – inverted terminal repeats of AAV. The nick sites of the origin are shown as yellow rectangles and the termini of RCT are marked by red rectangles. Dashed blue lines indicate nascent DNA strands and blue arrows show the direction of DNA synthesis. Black dotted arrows indicate nucleophilic attack of the catalytic tyrosine residue on the phosphodiester bond in the DNA or of the free 3′-OH DNA end on the phosphotyrosine bond between the enzyme and the DNA (adopted from Chandler et al., 2013; Thomas and Pritham, 2015; Grabundzija et al., 2016).

FIGURE 4

Multifunctional RCR initiator proteins encoded by different types of genetic elements.