The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. Synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Although the specific replication origin organization structure and recognition varies from species to species, some common characteristics are shared.

A key prerequisite for DNA replication is that it must occur with extremely high fidelity and efficiency exactly once per cell cycle to prevent the accumulation of genetic alterations with potentially deleterious consequences for cell survival and organismal viability. Incomplete, erroneous, or untimely DNA replication events can give rise to mutations, chromosomal polyploidy or aneuploidy, and gene copy number variations, each of which in turn can lead to diseases, including cancer. To ensure complete and accurate duplication of the entire genome and the correct flow of genetic information to progeny cells, all DNA replication events are not only tightly regulated with cell cycle cues but are also coordinated with other cellular events such as transcription and DNA repair. Additionally, origin sequences commonly have high AT-content across all kingdoms, since repeats of adenine and thymine are easier to separate because their base stacking interactions are not as strong as those of guanine and cytosine.

DNA replication is divided into different stages. During initiation, the replication machineries – termed replisomes – are assembled on DNA in a bidirectional fashion. These assembly loci constitute the start sites of DNA replication or replication origins. In the elongation phase, replisomes travel in opposite directions with the replication forks, unwinding the DNA helix and synthesizing complementary daughter DNA strands using both parental strands as templates. Once replication is complete, specific termination events lead to the disassembly of replisomes. As long as the entire genome is duplicated before cell division, one might assume that the location of replication start sites does not matter; yet, it has been shown that many organisms use preferred genomic regions as origins. The necessity to regulate origin location likely arises from the need to coordinate DNA replication with other processes that act on the shared chromatin template to avoid DNA strand breaks and DNA damage.

More than five decades ago, Jacob, Brenner, and Cuzin proposed the replicon hypothesis to explain the regulation of chromosomal DNA synthesis in E. coli. The model postulates that a diffusible, trans-acting factor, a so-called initiator, interacts with a cis-acting DNA element, the replicator, to promote replication onset at a nearby origin. Once bound to replicators, initiators (often with the help of co-loader proteins) deposit replicative helicases onto DNA, which subsequently drive the recruitment of additional replisome components and the assembly of the entire replication machinery. The replicator thereby specifies the location of replication initiation events, and the chromosome region that is replicated from a single origin or initiation event is defined as the replicon.

A fundamental feature of the replicon hypothesis is that it relies on positive regulation to control DNA replication onset, which can explain many experimental observations in bacterial and phage systems. For example, it accounts for the failure of extrachromosomal DNAs without origins to replicate when introduced into host cells. It further rationalizes plasmid incompatibilities in E. coli, where certain plasmids destabilize each other's inheritance due to competition for the same molecular initiation machinery. By contrast, a model of negative regulation (analogous to the replicon-operator model for transcription) fails to explain the above findings. Nonetheless, research subsequent to Jacob's, Brenner's and Cuzin's proposal of the replicon model has discovered many additional layers of replication control in bacteria and eukaryotes that comprise both positive and negative regulatory elements, highlighting both the complexity and the importance of restricting DNA replication temporally and spatially.

The concept of the replicator as a genetic entity has proven very useful in the quest to identify replicator DNA sequences and initiator proteins in prokaryotes, and to some extent also in eukaryotes, although the organization and complexity of replicators differ considerably between the domains of life. While bacterial genomes typically contain a single replicator that is specified by consensus DNA sequence elements and that controls replication of the entire chromosome, most eukaryotic replicators – with the exception of budding yeast – are not defined at the level of DNA sequence; instead, they appear to be specified combinatorially by local DNA structural and chromatin cues. Eukaryotic chromosomes are also much larger than their bacterial counterparts, raising the need for initiating DNA synthesis from many origins simultaneously to ensure timely replication of the entire genome. Additionally, many more replicative helicases are loaded than activated to initiate replication in a given cell cycle. The context-driven definition of replicators and selection of origins suggests a relaxed replicon model in eukaryotic systems that allows for flexibility in the DNA replication program. Although replicators and origins can be spaced physically apart on chromosomes, they often co-localize or are located in close proximity; for simplicity, we will thus refer to both elements as 'origins' throughout this review. Taken together, the discovery and isolation of origin sequences in various organisms represents a significant milestone towards gaining mechanistic understanding of replication initiation. In addition, these accomplishments had profound biotechnological implications for the development of shuttle vectors that can be propagated in bacterial, yeast and mammalian cells.

Most bacterial chromosomes are circular and contain a single origin of chromosomal replication (oriC). Bacterial oriC regions are surprisingly diverse in size (ranging from 250 bp to 2 kbp), sequence, and organization; nonetheless, their ability to drive replication onset typically depends on sequence-specific readout of consensus DNA elements by the bacterial initiator, a protein called DnaA. Origins in bacteria are either continuous or bipartite and contain three functional elements that control origin activity: conserved DNA repeats that are specifically recognized by DnaA (called DnaA-boxes), an AT-rich DNA unwinding element (DUE), and binding sites for proteins that help regulate replication initiation. Interactions of DnaA both with the double-stranded (ds) DnaA-box regions and with single-stranded (ss) DNA in the DUE are important for origin activation and are mediated by different domains in the initiator protein: a Helix-turn-helix (HTH) DNA binding element and an ATPase associated with various cellular activities (AAA+) domain, respectively. While the sequence, number, and arrangement of origin-associated DnaA-boxes vary throughout the bacterial kingdom, their specific positioning and spacing in a given species are critical for oriC function and for productive initiation complex formation.

Among bacteria, E. coli is a particularly powerful model system to study the organization, recognition, and activation mechanism of replication origins. E. coli oriC comprises an approximately ~260 bp region containing four types of initiator binding elements that differ in their affinities for DnaA and their dependencies on the co-factor ATP. DnaA-boxes R1, R2, and R4 constitute high-affinity sites that are bound by the HTH domain of DnaA irrespective of the nucleotide-binding state of the initiator. By contrast, the I, τ, and C-sites, which are interspersed between the R-sites, are low-affinity DnaA-boxes and associate preferentially with ATP-bound DnaA, although ADP-DnaA can substitute for ATP-DnaA under certain conditions. Binding of the HTH domains to the high- and low-affinity DnaA recognition elements promotes ATP-dependent higher-order oligomerization of DnaA's AAA+ modules into a right-handed filament that wraps duplex DNA around its outer surface, thereby generating superhelical torsion that facilitates melting of the adjacent AT-rich DUE. DNA strand separation is additionally aided by direct interactions of DnaA's AAA+ ATPase domain with triplet repeats, so-called DnaA-trios, in the proximal DUE region. The engagement of single-stranded trinucleotide segments by the initiator filament stretches DNA and stabilizes the initiation bubble by preventing reannealing. The DnaA-trio origin element is conserved in many bacterial species, indicating it is a key element for origin function. After melting, the DUE provides an entry site for the E. coli replicative helicase DnaB, which is deposited onto each of the single DNA strands by its loader protein DnaC.