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 multienzyme complexes, in which multiple catalytic domains are found in a single polypeptide chain. Protein ...

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 se ...

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 living organisms acting as the most essential part for biological inheritan ...

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

genome In the fields of molecular biology and genetics, 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 ...

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 series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...

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 (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were am ...

, 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

archaeal Archaea ( ; singular archaeon ) is a domain of single-celled organisms. These microorganisms lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebac ...

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 (ORC) protein,

Cdc6 Cell division control protein 6 homolog is a protein that in humans is encoded by the ''CDC6'' gene. The protein encoded by this gene is highly similar to Saccharomyces cerevisiae Cdc6, a protein essential for the initiation of DNA replication. ...

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 su ...

, and a homohexamer of the

minichromosome maintenance The minichromosome maintenance protein complex (MCM) is a DNA helicase essential for genomic DNA replication. Eukaryotic MCM consists of six gene products, Mcm2–7, which form a heterohexamer. As a critical protein for cell division, MCM is also t ...

(MCM) protein. ''Sulfolobus islandicus'' also uses a Cdt1 homologue to recognize one of its replication origins. The

eukaryotic Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacte ...

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

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'' (''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 the 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 three short claws o ...

'' (''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. The pre-RC is complete when DnaA occupies all of the high and low affinity 9-bp binding sites. 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 fungus microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have b ...

'' (''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 series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and subs ...

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 mRNA and proteins in preparation for subsequent steps ...

of the cell cycle when

cyclin-dependent kinase Cyclin-dependent kinases (CDKs) are the families of protein kinases first discovered for their role in regulating the cell cycle. They are also involved in regulating transcription, mRNA processing, and the differentiation of nerve cells. They ...

(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. 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 dnaC is a loading factor that complexes with the C-terminus of helicase dnaB and inhibits it from unwinding the dsDNA at a replication fork. A dnaB and dnaC associate near the dnaA bound origin for each of the ssDNA. One dnaB-dnaC complex is orie ...

helicase loader. Single-strand binding proteins stabilize the newly formed replication bubble and interact with the DnaG primase. 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 crea ...

III, and replication begins. In eukaryotes, MCM heterohexamer is phosphorylated by

CDC7 Cell division cycle 7-related protein kinase is an enzyme that in humans is encoded by the ''CDC7'' gene. The Cdc7 kinase is involved in regulation of the cell cycle at the point of chromosomal DNA replication. The gene CDC7 appears to be co ...

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 replisome is a complex molecular machine that carries out replication of DNA. The replisome first unwinds double stranded DNA into two single strands. For each of the resulting single strands, a new complementary sequence of DNA is synthe ...

; 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 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 protein complexes which degrade unneeded or damaged proteins by proteolysis, a chemical reaction that breaks peptide bonds. Enzymes that help such reactions are called proteases. Proteasomes are part of a major mechanism by whi ...

, 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 ...

via

phosphorylation In chemistry, phosphorylation is the attachment of a phosphate group to a molecule or an ion. This process and its inverse, dephosphorylation, are common in biology and could be driven by natural selection. Text was copied from this source, wh ...

. 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 blend of the Greek ''caeno-'' (recent), ''rhabditis'' (r ...

'', ''

Drosophila melanogaster ''Drosophila melanogaster'' is a species of fly (the taxonomic 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" or "pomace fly". Starting with ...

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

mammals Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur o ...

. 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 wherein an organism is exceptionally small, and mostly occurs in the animal kingdom. In humans, it is sometimes defined as an adult height of less than , regardless of sex; the average adult height among people with dw ...

, absent or hypoplastic patellae, small ears, impaired pre- and post-natal growth, and

microcephaly Microcephaly (from New 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 ...

. Known mutations are in the

ORC4 Origin recognition complex subunit 4 is a protein that in humans is encoded by the ORC4 (ORC4L) gene. Function The origin recognition complex (ORC) is a highly conserved six subunit protein complex essential for the initiation of the DNA repli ...

ORC6 Origin recognition complex subunit 6 is a protein that in humans is encoded by the ORC6 (ORC6L) gene. Background The origin recognition complex (ORC) is a highly conserved six subunit protein complex essential for the initiation of the DNA repl ...

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 Cell division control protein 6 homolog is a protein that in humans is encoded by the ''CDC6'' gene. The protein encoded by this gene is highly similar to Saccharomyces cerevisiae Cdc6, a protein essential for the initiation of DNA replication. ...

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