Specific mutations within the AT-rich region of a plasmid replication origin affect either origin opening or helicase loading

Abstract

Prokaryotic and eukaryotic replicons possess a distinctive region containing a higher than average number of adenine and thymine residues (the AT-rich region) where, during the process of replication initiation, the initial destabilization (opening) of the double helix takes place. In many prokaryotic origins, this region consists of repeated 13-mer motifs whose function has not yet been specified. Here we identify specific mutations within the 13-mer sequences of the broad-host-range plasmid RK2 that can result in defective origin opening or that do not affect opening but induce defects in helicase loading. We also show that after the initial recruitment of helicase at the DnaA-box sequences of the plasmid origin, the helicase is translocated to the AT-rich region in a reaction requiring specific sequence of the 13-mers and appropriate facing of the origin motifs. Our results demonstrate that specific sequences within the AT-rich region of a replication origin are required for either origin opening or helicase loading.13-mer sequences ͉ DNA replication ͉ DnaB helicase ͉ plasmid RK2 T he initiation of DNA synthesis requires a mechanism to coordinate DNA unwinding, helicase recruitment, and sitespecific helicase loading. Binding of the initiator (Rep), a single protein, or a complex of several proteins to the replication origin results in the melting of the double-stranded DNA helix in a region of the origin with low internal thermodynamic stability because of a higher than average content of adenine and thymine residues (AT-rich region). In plasmid replicons, the involvement of both the host-encoded replication initiator DnaA and the plasmid encoded Rep protein is essential for origin melting, helicase recruitment, and helicase loading onto the ssDNA in the opened AT-rich region.DNA replication of the broad-host-range plasmid RK2 is initiated by the binding of the plasmid-encoded protein TrfA to direct repeats (iterons) present at its replication origin (oriV), a process similar to that of other plasmid systems (1, 2). The formation of an open complex at oriV by the TrfA initiation protein requires HU and is stabilized by the host DnaA protein (3). In contrast, the initiation of replication at the Escherichia coli chromosomal origin, oriC, involves a single initiator, DnaA, binding to DnaA-box sequences and resulting in duplex DNA melting (4-7). During replication initiation at RK2 oriV, helix destabilization occurs within the AT-rich region, which includes four 13-mer repeated sequences (L, M1, M2, R) that exhibit a DNA consensus sequence of TAAACnTTnTTTT (3). Similar motifs have been identified in a majority of theta-replicating plasmids and bacterial chromosomes including E. coli, where three 13-mers (L, M and R) have been identified in oriC (4-6). The position of the 13-mer clusters and their specific sequences are important for proper functioning of both oriV (8, 9) and oriC (5, 10, 11). Although the importance of 13-mers is well established, their exact role is not completely understood.Certain E. coli pro...

AT-rich region and repeated sequences – the essential elements of replication origins of bacterial replicons

“…The iterons in plasmid origins are clustered similarly as the DnaA-boxes in chromosomal origins, on one side of an AT-rich DUE ( Fig. Both in vivo and in vitro tests revealed that specific alterations within the 13-mer sequences result in inhibition of the RK2 oriV opening and consequently in the lack of origin activity (Kowalczyk et al, 2005;Rajewska et al, 2008). The open complex formation at the plasmid origins is induced by Rep protein binding to the iterons; however, some plasmids still require DnaA to stabilize or enhance the origin unwinding (Kawasaki et al, 1996;Park et al, 1998;Kruger et al, 2001).…”

“…The DnaA and DnaC interactions with the ssDNA within the open AT-rich region are demonstrated for chromosomal origins (see paragraph above). Specific alterations within the 13-mer sequences, which do no affect origin opening, significantly disturb the E. coli DnaB helicase translocation from the DnaA-boxes to the RK2 AT-rich region (Rajewska et al, 2008). During the RK2 plasmid replication initiation, the E. coli DnaB helicase complex consisting of DnaA and DnaC proteins was shown at dsDNA DnaA-box repeats located outside the AT-rich region of the plasmid origin Pacek et al, 2001).…”

Two replication initiators – one mechanism for replication origin opening?

“…At the RK2 plasmid origin, the DnaBC-complex is bound to DnaAboxes by way of its interaction with DnaA (Pacek et al, 2001). TrfA then activates DnaB helicase and translocates it to the AT-rich region (Konieczny and Helinski, 1997;Rajewska et al, 2008). Different mechanisms have been observed for the DnaB recruitment process as utilization of specific replication proteins is host dependent.…”

“…It is located 60 bp downstream of the iterons and contains four consensus 13-nucleotide repeat sequences: L, M1, M2, R. Three of them (L, M1, R) have the same orientation, while M2 has an opposite one. The position of these sequences is critical for origin opening and helicase loading during assembly of the replication complex (Doran et al, 1998;Konieczny et al, 1997;Kowalczyk et al, 2005;Rajewska et al, 2008Rajewska et al, , 2012.…”

MAGNITUDE OF THYMINE IN DIFFERENT FRAMES OF MESSENGER RNAs

“…There is record of evidences state that specific mutations within the AT-rich region of replication origin affect either origin opening or helicase loading [5]. This is both in prokaryotic and eukaryotic replicons.…”