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Read this article to learn about the three phases of DNA replication process.
The three phases of replication process are: (1) Initiation (2) Elongation and (3) Termination.
Replication in prokaryotes and eukaryotes occurs by very similar mechanisms, and thus most of the information presented here for bacterial replication applies to eukaryotic cells as well.
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It is composed of three phases which are listed below:
(a) Initiation:
It involves recognition of the positions on a DNA molecule where replication will begin.
(b) Elongation:
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It includes the events occurring at the replication fork, where the parent poly-nucleotides are copied.
(c) Termination:
It is less understood. It occurs when the parent molecule has been completely replicated.
(a) Initiation:
In a cell, DNA replication begins at specific locations in the genome, called “origins”. In case of E. coli the origin of replication is a sequence of approximately 245 base pairs (bp) called oriC. Origins contain DNA sequences recognized by replication initiator proteins (e.g. DnaA in E. coli and the Origin Recognition Complex in yeast), these proteins bind to start the process of replication. The initiator proteins recruit other proteins to separate the two strands and initiate replication forks.
Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. The replication fork is a structure which forms when DNA is being replicated. It is created through the action of helicase, which breaks the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching “prongs”, each one made up of a single strand of DNA.
In bacteria, which have a single origin of replication on their circular chromosome, this process eventually creates a “theta structure” (resembling the Greek letter theta: 8). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins and whose replication forks progress for shorter distances. For example yeast has about 322 origins, which corresponds to 1 origin per 36 kb of DNA, and humans have some 20,000 origins, or 1 origin for every 150 kb of DNA. Once initiated, two replication forks can emerge from the origin and progress in opposite direction along the DNA. Replication is therefore bidirectional with most genomes (Fig. 3.4).
Initiation of DNA Replication in Microorganisms (E. coli):
We know substantially more about DNA synthesis in prokaryotes than in eukaryotes. As we have discussed that oriC of E.coli spans 245 bp of DNA. Sequence analysis of this segment shows that it contains two short repeat motifs, one of nine nucleotides and the other of 13 nucleotides. The five copies of nine nucleotide repeat motif are presented dispersedly throughout oriC.
These regions are the binding site for a protein called DnaA. As there are five copies of the binding sequences, it might be imagined that five copies of DnaA attach to the origin, but in fact bound DnaA proteins cooperate with unbound molecules until some 30 copies are associated with the origin. Attachment occurs only when the DNA is negatively super-coiled, as is the normal situation for the E. coli chromosome.
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The result of DnaA binding is that the double helix opens up (melts) within the tandem array of three AT-rich, 13 nucleotide repeats located at one end of the oriC sequence. The exact mechanism is unknown but DnaA does not appear to possess the enzymatic activity needed to break base pairs, and it is therefore assumed that the helix is melted by torsional stresses introduced by attachment of the DnaA proteins.
An attractive model imagines that the DnaA proteins form a barrel-like structure around which the helix is wound. Melting the helix is promoted by HU, the most abundant of the DNA packaging proteins of E. coli. Melting of the helix initiates a series of events that construct a new replication fork at either end of the open region. The first step is the attachment of a pre-priming complex at each of these two positions.
Each pre-priming complex initially comprises 12 proteins, six copies of DnaB, and six copies of DnaC, but DnaC has a transitory role and is released from the complex soon after it is formed, its function probably being simply to aid the attachment of DnaB.
The latter is a helicase, an enzyme which can break base pairs. DnaB begins to increase the single-stranded region within the origin, enabling the enzymes involved in the elongation phase of replication in E. coli as the replication forks now start to progress away from the origin and DNA copying begins.
Initiation of DNA Replication in Yeast:
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Origins identified in yeast are called autonomously replicating sequences, or ARSs. Atypical yeast origin is shorter than E. coli oriC, being usually less than 200 bp in length. In it four sub-domains are recognized.
Two of these sub-domains A and B1 – make up the origin recognition sequence, a stretch of some 40 bp in total that is the binding site for the Origin recognition complex (ORC), a set of six proteins that attach to the origin.
ORCs have been described as yeast versions of the E. coli DnaA proteins, but this interpretation is probably not strictly correct because ORCs appear to remain attached to yeast origins throughout the cell cycle. Rather these are genuine initiator proteins. It is more likely that ORCs are involved in the regulation of genome replication, acting as mediators between replication origins and the regulatory signals that coordinate the initiation of DNA replication with the cell cycle.
There are similar sequences in yeast to that of oriC of E. coli. This leads us to the two other conserved sequences in the typical yeast origin, sub-domains B2 and B3. Our current understanding suggests that these two sub-domains function in a manner similar to the E. coli origin. Sub-domain B2 appears to correspond to the 13-nucleotide repeat array of the E. coli origin, being the position at which the two strands of the helix are first separated.
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This melting is induced by torsional stress introduced by attachment of a DNA-binding protein, ARS binding factor 1 (ABF1), which attaches to sub-domain B3. As in E. coli, melting of the helix within a yeast replication origin is followed by attachment of the helicase and other replication enzymes to the DNA, completing the initiation process and enabling the replication forks to begin their progress along the DNA. Replications origins in higher eukaryotes have not been much understood.
(b) Elongation:
Once replication has been initiated; the replication forks progress along the DNA and participate in the synthesis of new strand. At the chemical level, the template dependent synthesis of DNA is very similar to the template-dependent synthesis of RNA that occurs during transcription, but the two processes are quite different.
1. Discontinuous strand synthesis and the priming problem- During DNA replication both strands of the double helix must be copied. However, DNA polymerase enzymes are only able to synthesize DNA in the 5′ 3′ direction. This means that one strand of the parent double helix, called the leading strand, can be copied out in a continuous manner, but replication of the lagging strand has to be carried out in a discontinuous fashion, resulting in a series of short segments that must be ligated together to produce the intact daughter strand.
These short segments of poly-nucleotides are called as Okazaki fragments. These fragments were first isolated from E. coli bacteria in 1969. Okazaki fragments are 1000-2000 nucleotides in length, but in eukaryotes the equivalent fragments appear to be much shorter, perhaps less than 200 nucleotides in length.
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2. The another feature of DNA replication is that DNA polymerase cannot initiate DNA synthesis on a molecule that is entirely single stranded: there must be short single stranded region to provide a 3′ end onto which the enzyme can add new nucleotides. Nucleotides are added at a rate of 50,000 bases per minute. The choice of nucleotide is determined by complementary nature.
At this rate chances of error are one in one thousand base pair replicated. However, actual rate is quite low (one in one billion). This is equal to about one error per genome per one thousand bacterial replication cycles. This error is further corrected by proofreading (Removal of mismatch nucleotide by DNA polymerase III). This means that primers are needed, one to initiate complementary strand synthesis on the leading polynucleotide, and one for every segment of discontinuous DNA synthesized on the lagging strand.
As DNA polymerase cannot deal with an entirely single stranded template, RNA polymerases have no difficulty in this respect, so the primers for DNA replication are made of RNA. In bacteria, primers are synthesized by primase, a special RNA polymerase with each primer being 4-15 nucleotides in length and most starting with the sequence 5′-AG-3′. Once the primer has been completed, strand synthesis is continued by DNA polymerase III.
In eukaryotes the situation is more complex because the primase is tightly bound to DNA polymerase a, and cooperates with this enzyme in synthesis of the first few nucleotides of a new polynucleotide. This primase synthesizes an RNA primer of 8-12 nucleotides, and then hands over to DNA polymerase a, which extends the RNA primer by adding about 20 nucleotides of DNA. After completion of DNA-RNA primer, DNA synthesis is continued by the main replicative enzyme, DNA polymerase 5.
Priming needs to occur just once on the leading strand, within the replication origin, because once primed, the leading-strand copy is synthesized continuously until replication is completed. On the lagging strand, priming is a repeated process that must occur every time a new Okazaki fragment is initiated.
3. Replication fork elongation-As with the attachment of DnaB helicase, followed by extension of the melted region of the replication origin, the initiation phase ends. After the helicase has bound to the origin to form pre-priming complex, the primase is involved, resulting in the primosome, which initiates replication of the leading strand. It does this by synthesizing the RNA primer that DNA polymerase III needs in order to begin copying the template. More than one helicase is known and this enzyme is involved in various processes, such as transcription, recombination besides replications.
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4. Complementary strands of a DNA tend to become duplex. During the process of replication, these sticky single stranded DNA are prevented to become duplex by special proteins called as single strand binding proteins (SSBs). Once 1000-2000 nucleotides are added in the leading strand, synthesis of lagging strand or Okazaki fragments began. This also requires an RNA primer and DNA polymerase III similar to leading strand.
5. DNA polymerase I is involved in removing the RNA primer from Okazaki fragments, having 5′ → 3′ exo-nuclease activity. Gap created by primer is filled by adding nucleotides at 3′ end. The nick between two Okazaki fragments is sealed by DNA ligase by the formation of phosphodiester bonds (Fig. 3.5).
(c) Termination:
Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome. E coli regulate this process through the use of termination sequences which, when bound by the Tus protein, enable only one direction of replication fork to pass through.
As a result, the replication forks are constrained to always meet within the termination region of the chromosome. Eukaryotes initiate DNA replication at multiple points in the chromosome, so replication forks meet and terminate at many points in the chromosome; these are not known to be regulated in any particular manner.
