A pre-replication complex (pre-RC) is a

protein complex A protein complex or multiprotein complex is a group of two or more associated polypeptide chains. Protein complexes are distinct from multidomain enzymes, in which multiple active site, catalytic domains are found in a single polypeptide chain. ...

that forms at the

origin of replication 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 semi ...

during the initiation step of

DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all life, living organisms, acting as the most essential part of heredity, biolog ...

. Formation of the pre-RC is required for DNA replication to occur. Complete and faithful replication of the

genome A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...

ensures that each daughter cell will carry the same genetic information as the parent cell. Accordingly, formation of the pre-RC is a very important part of the

cell cycle The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell (biology), cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA (DNA re ...

Components

As organisms evolved and became increasingly more complex, so did their pre-RCs. The following is a summary of the components of the pre-RC amongst the different domains of life. In

bacteria Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one Cell (biology), biological cell. They constitute a large domain (biology), domain of Prokaryote, prokaryotic microorganisms. Typically a few micr ...

, the main component of the pre-RC is DnaA. The pre-RC is complete when DnaA occupies all of its binding sites within the bacterial origin of replication ( oriC). The particular sites on the oriC that DnaA binds to determines if the cell has a bORC (bacterial Origin Recognition Complex) or a pre-RC. The archaeal pre-RC is very different from the bacterial pre-RC and can serve as a simplified model of the eukaryotic pre-RC. It is composed of a single

origin recognition complex In molecular biology, origin recognition complex (ORC) is a multi-subunit DNA binding complex (6 subunits) that binds in all eukaryotes and archaea in an Adenosine triphosphate, ATP-dependent manner to origins of replication. The subunits of this ...

(ORC) protein, Cdc6/

ORC1 Origin recognition complex subunit 1 is a protein that in humans is encoded by the ORC1 gene. It is closely related to CDC6, and both are the same protein in archaea. Function The origin recognition complex (ORC) is a highly conserved six s ...

, and a homohexamer of the minichromosome maintenance (MCM) protein. ''Sulfolobus islandicus'' also uses a Cdt1 homologue to recognize one of its replication origins. The

eukaryotic The eukaryotes ( ) constitute the Domain (biology), domain of Eukaryota or Eukarya, organisms whose Cell (biology), cells have a membrane-bound cell nucleus, nucleus. All animals, plants, Fungus, fungi, seaweeds, and many unicellular organisms ...

pre-RC is the most complex and highly regulated pre-RC. In most eukaryotes it is composed of six ORC proteins (ORC1-6), Cdc6,

Cdt1 CDT1 (Chromatin licensing and DNA replication factor 1) is a protein that in humans is encoded by the ''CDT1'' gene. It is a licensing factor that functions to limit DNA from replicating more than once per cell cycle. Role in pre-replication co ...

, and a heterohexamer of the six MCM proteins (MCM2-7). The MCM heterohexamer arguably arose via MCM gene duplication events and subsequent divergent evolution. The pre-RC of ''

Schizosaccharomyces pombe ''Schizosaccharomyces pombe'', also called "fission yeast", is a species of yeast used in traditional brewing and as a model organism in molecular and cell biology. It is a unicellular eukaryote, whose cells are rod-shaped. Cells typically meas ...

'' (''S. pombe'') is notably different from that of other eukaryotes; Cdc6 is replaced by the homologous Cdc18 protein. Sap1 is also included in the ''S. pombe'' pre-RC because it is required for Cdc18 binding. The pre-RC of ''

Xenopus laevis The African clawed frog (''Xenopus laevis''), also known as simply xenopus, African clawed toad, African claw-toed frog or the ''platanna'') is a species of African aquatic frog of the family Pipidae. Its name is derived from the short black ...

'' (''X. laevis'') also has an additional protein, MCM9, which helps load the MCM heterohexamer onto the origin of replication. The structure of the ORC, MCM, as well as the intermediate ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex has been resolved.

Recognition of the origin of replication

Recognition of the origin of replication is a critical first step in the formation of the pre-RC. In different domains of life this process is accomplished differently. In prokaryotes, origin recognition is accomplished by DnaA. DnaA binds tightly to a 9-base pair consensus sequence in oriC; 5' – TTATCCACA – 3'. There are 5 such 9-bp sequences (R1-R5) and 4 non-consensus sequences (I1-I4) within oriC that DnaA binds with differential affinity. DnaA binds R4, R1, and R2 with high affinity and R5, I1, I2, I3, and R3 with lesser affinity. ''In vivo,'' it has been observed that the DnaA binding to recognition sites occurs in the order: R1, R2, then R4, which forms the bORC. Afterwards, the other lower affinity, 9 bp recognition sites bind to DnaA, which forms the pre-RC. Archaea have 1–3 origins of replication. The origins are generally AT-rich tracts that vary based on the archaeal species. The singular archaeal ORC protein recognizes the AT-rich tracts and binds DNA in an ATP-dependent fashion. Eukaryotes typically have multiple origins of replication; at least one per chromosome. ''

Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungal microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have be ...

'' (''S. cerevisiae'') is the only known eukaryote with a defined initiation sequence TTTTTATG/ATTTA/T. This initiation sequence is recognized by ORC1-5. ORC6 is not known to bind DNA in ''S. cerevisiae''. Initiation sequences in ''S. pombe'' and higher eukaryotes are not well defined. However, the initiation sequences are generally either AT-rich or exhibit bent or curved DNA topology. The ORC4 protein is known to bind the AT-rich portion of the origin of replication in ''S. pombe'' using AT hook motifs. The mechanism of origin recognition in higher eukaryotes is not well understood but it is thought that the ORC1-6 proteins depend on unusual DNA topology for binding.

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Overview of chromosome duplication in the cell cycle

Assembly of the pre-replication complex only occurs during late

M phase The cell cycle, or cell-division cycle, is the sequential series of events that take place in a cell that causes it to divide into two daughter cells. These events include the growth of the cell, duplication of its DNA (DNA replication) and ...

and early

G1 phase The G1 phase, gap 1 phase, or growth 1 phase, is the first of four phases of the cell cycle that takes place in eukaryotic cell division. In this part of interphase, the cell synthesizes Messenger RNA, mRNA and proteins in preparation for subsequ ...

of the cell cycle when

cyclin-dependent kinase Cyclin-dependent kinases (CDKs) are a predominant group of serine/threonine protein kinases involved in the regulation of the cell cycle and its progression, ensuring the integrity and functionality of cellular machinery. These regulatory enzym ...

(CDK) activity is low. This timing and other regulatory mechanisms ensure that DNA replication will only occur once per cell cycle. Assembly of the pre-RC relies on prior origin recognition, either by DnaA in prokaryotes or by ORC in archaea and eukaryotes. The pre-RC of prokaryotes is complete when DnaA occupies all possible binding sites within the oriC. DnaA can only bind to the low affinity sites on the oriC once the protein fis is removed from the oriC. Removal of fis, the protein IHF (integrated host factor) binds to a site between R1 and R2, which allows DnaA to bind to the low affinity sites on the oriC. This completes the pre-RC. The pre-RC of archaea requires ORC binding of the origin. After this, Cdc6 and the MCM homohexameric complex bind in a sequential fashion. Eukaryotes have the most complex pre-RC. After ORC1-6 bind the origin of replication, Cdc6 is recruited. Cdc6 recruits the licensing factor Cdt1 and MCM2-7. Cdt1 binding and ATP hydrolysis by the ORC and Cdc6 load MCM2-7 onto DNA. There is a stoichiometric excess of the MCM proteins over the ORC and Cdc6 proteins, indicating that there may be multiple MCM heterohexamers bound to each origin of replication.

Initiation of replication

After the pre-RC is formed it must be activated and the replisome assembled in order for DNA replication to occur. In prokaryotes, DnaA hydrolyzes ATP in order to unwind DNA at the oriC. This denatured region is accessible to the DnaB helicase and DnaC helicase loader. Single-strand binding proteins stabilize the newly formed replication bubble and interact with the DnaG

primase DNA primase is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA (or DNA in some living organisms) segment called a primer complementary to a ssDNA (single-stranded ...

. DnaG recruits the replicative

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...

III, and replication begins. In eukaryotes, MCM heterohexamer is phosphorylated by CDC7 and CDK, which displaces Cdc6 and recruits MCM10. MCM10 cooperates with MCM2-7 in the recruitment of Cdc45. Cdc45 then recruits key components of the replisome; the replicative DNA polymerase α and its primase. DNA replication can then begin.

Prevention of pre-replication complex re-assembly

During each cell cycle, it is important that the genome be completely replicated once and only once. Formation of the pre-replication complex during late M and early G1 phase is required for genome replication, but after the genome has been replicated the pre-RC must not form again until the next cell cycle. In prokaryotes, various studies have demonstrated that the pre-RC is a complex that is only present for a fraction of the cell cycle. Once a cellular division occurs, the pre-RC must revert back to the bORC to ensure that only one round of DNA replication occurs during division. In ''E. coli'', there are 11 GATC sites in the oriC that undergo hemimethylation during DNA replication. The protein SeqA binds to these sites preventing remethylation and blocking the binding of DnaA to low affinity sites for approximately one third of the cell cycle. However, SeqA does not block DnaA from binding to the R1, R2, and R4 sites. Thus, the bORC is reset and is prepared to undergo another conversion to the pre-RC. In S. cerevisiae, CDKs prevent formation of the replication complex during late G1, S, and G2 phases by excluding MCM2-7 and Cdt1 from the nucleus, targeting Cdc6 for degradation by the

proteasome Proteasomes are essential protein complexes responsible for the degradation of proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases. Proteasomes are found inside all e ...

, and dissociating ORC1-6 from

chromatin Chromatin is a complex of DNA and protein found in eukaryote, eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important r ...

via

phosphorylation In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writ ...

. Prevention of re-replication in S. pombe is slightly different; Cdt1 is degraded by the proteasome instead of merely being excluded from the nucleus. Proteolytic regulation of Cdt1 is shared by higher eukaryotes including ''

Caenorhabditis elegans ''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a Hybrid word, blend of the Greek ''caeno-'' (recent), ''r ...

'', ''

Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (an insect of the Order (biology), order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly", "pomace fly" ...

'', ''X. laevis'', and

mammals A mammal () is a vertebrate animal of the class Mammalia (). Mammals are characterised by the presence of milk-producing mammary glands for feeding their young, a broad neocortex region of the brain, fur or hair, and three middle e ...

. Metazoans have a fourth mechanism to prevent re-replication; during S and G2 geminin binds to Cdt1 and inhibits Cdt1 from loading MCM2-7 onto the origin of replication.

Meier-Gorlin syndrome

Defects in components of the eukaryotic replication complex are known to cause Meier-Gorlin syndrome, which is characterized by

dwarfism Dwarfism is a condition of people and animals marked by unusually small size or short stature. In humans, it is sometimes defined as an adult height of less than , regardless of sex; the average adult height among people with dwarfism is . '' ...

, absent or

hypoplastic Hypoplasia (; adjective form ''hypoplastic'') is underdevelopment or incomplete development of a Tissue (biology), tissue or Organ (biology), organ.patellae, small ears, impaired pre- and post-natal growth, and

microcephaly Microcephaly (from Neo-Latin ''microcephalia'', from Ancient Greek μικρός ''mikrós'' "small" and κεφαλή ''kephalé'' "head") is a medical condition involving a smaller-than-normal head. Microcephaly may be present at birth or it m ...

. Known mutations are in the

, ORC4, ORC6,

CDT1 CDT1 (Chromatin licensing and DNA replication factor 1) is a protein that in humans is encoded by the ''CDT1'' gene. It is a licensing factor that functions to limit DNA from replicating more than once per cell cycle. Role in pre-replication co ...

, and CDC6 genes. The disease phenotype probably originates from reduced ability of cells to proliferate, leading to cell number, and general growth failure.

References

{{DNA replication DNA replication