6 - DNA Replication

When James Watson and Francis Crick determined the structure of the DNA double helix, they noticed that the structure provided clues to how DNA is copied prior to cell division. This copying process is called DNA replication (see figure 6.1).

Overview of DNA Replication

6.0.1_Overview_of_DNA_Replication.jpgFigure 6.1 Overview of DNA Replication --- Image created by SL

Watson and Crick proposed that during DNA replication, the two original DNA strands within the double helix separate, and two new strands of DNA are synthesized. The two original DNA strands are called template DNA strands or parental DNA strands; each of the newly synthesized DNA strands is called a daughter DNA strand.

When DNA nucleotides (deoxyribonucleoside triphosphates or dNTPs) are used to generate the daughter DNA strands, the AT/GC rule is followed. Hydrogen bonds are formed between the nitrogenous bases within the incoming nucleotides and the template strand nitrogenous bases. Then a phosphodiester bond is formed between the free 5’ phosphate on the incoming nucleotide and the free 3’ hydroxyl group on the growing daughter DNA strand. The dNTPs used as the substrates for DNA synthesis include deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP).

Key Questions

A. DNA Replication in Bacteria

Origin of Replication in Bacteria

The site on the bacterial chromosome where DNA replication begins is the origin of replication (see figure 6.2). The bacterium E. coli has a single origin of replication called OriC. OriC is a 275 base pair (bp)-long region that contains important DNA sequences, including:

DNA replication begins at OriC and proceeds in both directions (clockwise and counterclockwise) around the circular bacterial chromosome (bidirectional replication).  Further, a replicon is defined as all of the DNA replicated from a single origin. Since the entire E. coli chromosome is replicated from a single origin, the chromosome is one replicon.

6.2_OriC_in_E.Coli.jpgFigure 6.2 OriC in E.coli --- Image created by KMD

Key Questions

Replication Initiation

The steps involved in DNA replication in bacteria are (see figure 6.3):

  1. DnaA proteins bind to the DnaA box sequences. When DnaA proteins bind to ATP, DnaA binds tightly to the DnaA box sequences within OriC.
  2. The origin forms a loop and the individual DNA strands separate. Multiple copies of the DnaA bind to each other, forming a loop in the DNA.  The DNA loop promotes DNA strand separation within the AT-rich sequences of OriC. This looping of the DNA and strand separation requires ATP cleavage by the DnaA protein. After ATP is cleaved, the DnaA proteins are released from OriC.
  3. A copy of DNA helicase binds to each of the two separated DNA strands.
  4. The DNA helicases move along the template DNA strands, separating the DNA strands to form two replication forks. Template DNA strand separation starts at OriC and moves in both directions around the circular bacterial chromosome. DNA helicase cleaves ATP and uses the released energy to catalyze DNA strand separation.
  5. Single-stranded DNA binding proteins (SSBPs) bind to the separated single-stranded template DNA strands. SSBPs prevent the template DNA strands, separated by DNA helicase, from reforming hydrogen bonds, so that DNA replication can proceed.
EDI_6.3_Replication_Initiation_in_Bacteria_1-01.jpgFigure 6.3 Replication Initiation in Bacteria --- Image created by SL

Coordinating DNA Replication with Cell Division

Most bacteria divide quickly; for example, the cell division time of E. coli is approximately 20 minutes. If DNA replication in E. coli does not keep up with the division of the cytoplasm, daughter cells will be formed that lack chromosomes. On the other hand, if DNA replication occurs too quickly, daughter E. coli cells would contain more than one copy of the chromosome.

How is DNA replication and division of the cytoplasm coordinated? E. coli coordinates these two processes by regulating how often DNA replication starts. There are two general ways to regulate the initiation of DNA replication:

Key Questions

Replication Elongation

The elongation stage of DNA replication in bacteria consists of the following steps (see figure 6.4):

  1. RNA primers are synthesized. After the template DNA strands have separated, small RNA strands (10-12 nucleotides long) are synthesized that form hydrogen bonds with the template DNA strands. These RNA primers provide the free 3’-OH groups required by DNA polymerases to initiate daughter DNA strand synthesis.
     
  2. DNA synthesis occurs by reading the template DNA strands. Daughter DNA strands are synthesized in the 5’ to 3’ direction by adding dNTPs to free 3'-OH groups. However, because the template DNA strands are antiparallel to the daughter DNA strands, DNA polymerases read the template DNA strands in the 3’ to 5’ direction as the daughter DNA strands are synthesized. Note that as the DNA polymerase reads the template DNA strand 3' to 5' and synthesizes the daughter DNA strand 5' to 3', the DNA polymerase is moving in a single direction. 
     
    Since DNA polymerases only synthesize the daughter DNA strands in the 5’ to 3’ direction, the two daughter DNA strands synthesized at each replication fork are made in opposite directions. One newly synthesized daughter DNA strand is called the leading strand. The leading strand is synthesized in the same direction that the replication fork is moving as the template DNA strands are separated. The leading DNA strand requires only one RNA primer and DNA synthesis is continuous. The other newly synthesized daughter DNA strand at each replication fork is the lagging strand. The lagging strand is synthesized as a series of Okazaki fragments (1000–2000 nucleotide-long DNA fragments) in the opposite direction the replication fork is separating the template DNA strands. Each Okazaki fragment is initiated by a single RNA primer; the lagging DNA strand is synthesized in a discontinuous (fragmented) manner.
     
  3. The RNA primers are removed. Removing the RNA primers results in a gap between each Okazaki fragment.
     
  4. DNA synthesis fills the gaps left by the removed RNA primers.  DNA synthesis to fill the primer gaps occurs 5' to 3'.
     
  5. The adjacent Okazaki fragments are linked (ligated) together. Ligation of the adjacent Okazaki fragments forms a continuous lagging DNA strand.
EDI_6.4_Replication_Elongation-01_1.jpgFigure 6.4 Replication Elongation ---- Image by Genomics Education Programme. Image licesensed under CC BY 2.0

Key Questions

Proteins Involved in Elongation

The following proteins are involved in the elongation stage of DNA replication in bacteria (see figure 6.5):

EDI_6.5_Bacterial_Replication_Proteins-01-01_1.jpgFigure 6.5 Bacterial Replication Proteins ---  This image is used  from OpenStax (access for free at https://openstax.org/books/biology-2e/pages/1-introduction)

Key Questions

DNA Polymerase III Holoenzyme

DNA polymerase III is a holoenzyme (multi-protein enzyme complex) composed of at least ten unique protein types (see figure 6.6). Moreover, each of these unique protein types within the DNA polymerase III holoenzyme is present in multiple copies, making the overall composition of the DNA polymerase III holoenzyme quite complex. The protein subunit composition of the DNA polymerase III holoenzyme is as follows:

6.6_DNA_Polymerase_III_Holoenzyne.jpgFigure 6.6 DNA Polymerase III Holoenzyme.  The direction the replication fork is moving is shown by the arrow in the center of the image (i.e. the replication fork is moving from top to bottom)  The protein subunits on the left side of the image synthesize the lagging DNA strand; the protein subunits on the right synthesize the leading strand.  The α subunits (green), β subunits (orange), ε subunits (pink), and accessory subunits (tan) are indicated. --- Image created by SL

Key Questions

DNA Replication Proteins form Complexes

Many of the DNA replication enzymes described above are not physically separated. Each enzyme has a distinct function in DNA replication; however, many of these enzymes are physically linked to each other to form multiprotein “machines.” For example, the primosome is a protein complex formed by DNA helicase and DNA primase. The primosome moves along the DNA separating the DNA strands and simultaneously synthesizing lagging strand RNA primers.  Further, the primosome itself is part of a larger multi-subunit complex called the replisome. The replisome includes:

There is a single replisome per replication fork in the bacterium E. coli.  Since a replicating bacterial chromosome has two replication forks, there are two replisomes per bacterial chromosome.

Key Questions

DNA Polymerases in Bacteria

In the bacterium E. coli, there are five DNA polymerase types. We will focus our attention on DNA polymerases I and III, as these two enzymes are involved in DNA replication. The other three DNA polymerases (DNA polymerase II, IV, and V) are involved in repairing bacterial DNA that has been damaged by environmental agents.

DNA polymerase III (also called the DNA polymerase III holoenzyme; see above) replicates the leading and lagging DNA strands (has 5’ to 3’ polymerase activity). DNA polymerase III also contains a proofreading activity that removes DNA replication mistakes in the 3' to 5' direction (the so-called 3’ to 5’ exonuclease activity; see below).  DNA polymerase I is composed of a single protein subunit and functions to remove Okazaki fragment RNA primers in the 5' to 3' direction (i.e., the 5’ to 3’ exonuclease activity).  DNA polymerase I also fills in the gaps left by the removed RNA primers with DNA via its 5’ to 3’ polymerase activity and has 3’ to 5’ exonuclease activity (proofreading activity; see below).

All DNA polymerases have two unique features.  First, DNA polymerases require a free 3’-OH group provided by the primer to begin DNA synthesis. The primer used within cells is RNA; however, DNA polymerases can use DNA primers to synthesize DNA as well. In fact, DNA primers are commonly used when synthesizing DNA in the lab (see Part 8). Second, DNA polymerases synthesize the growing daughter strand in the 5’ to 3’ direction only.

Key Questions

DNA Polymerase Mechanism

DNA polymerases use the chemical energy stored within the high energy phosphate bonds of deoxyribonucleoside triphosphate (dNTP) molecules to synthesize the daughter DNA strands. Specifically, the DNA polymerase mechanism involves (see figure 6.7):

  1. The DNA polymerase reads a nitrogenous base in the template DNA strand and binds to the complementary dNTP according to the AT/GC rule. The incoming dNTP forms hydrogen bonds with the nitrogenous base in the template DNA strand.
  2. The free 3’-OH group on the growing daughter DNA strand reacts with the phosphate groups on the incoming dNTP.
  3. A high energy bond within the dNTP is broken releasing two of the phosphate groups in the form of pyrophosphate (PPi).
  4. The released energy is used to synthesize a new phosphodiester bond between the 3’ end of the growing DNA strand and the 5’ end of the incoming nucleotide.

The DNA polymerase III holoenzyme is processive. Processivity means that the DNA polymerase III holoenzyme can add many nucleotides to a daughter DNA strand without falling off the template DNA strand. This processivity is due to the four β subunits (sliding clamps; see above) found within the DNA polymerase III holoenzyme.

6.7_DNA_Polymerase_Mechanism.jpgFigure 6.7 DNA Polymerase Mechanism --- Image created by Michal Sobkowski and is licensed under CC BY 3.0.

Key Questions

Proofreading by DNA Polymerases

DNA polymerases incorporate the wrong nucleotide (i.e., a nucleotide that forms base pairs that deviate from the AT/GC rule) into a daughter DNA strand rarely. For example, the DNA polymerase III holoenzyme is thought to incorporate the wrong nitrogenous base once in every 10–100 million nitrogenous bases in a daughter DNA strand. This accuracy during DNA synthesis is called fidelity; both DNA polymerase I and the DNA polymerase III holoenzyme are said to have high fidelity (low error rates). The fidelity of DNA polymerases is the combination of three factors:

6.8_Proofreading.jpgFigure 6.8 Proofreading --- Image created by SL

Key Questions

Termination of Replication in Bacteria

DNA replication in E. coli  terminates at specific locations within the circular chromosome called termination (ter) sequences. Since there are two replication forks moving in opposite directions around the circular chromosome, there are two ter  DNA sequences.  Each ter sequence (the T1 and T2 sequences) stops the advancement of one of the two replication forks (see figure 6.9). Proteins called termination utilization substances (Tus) bind to the T1 and T2 sequences. Tus proteins release the replisomes from the two replication forks, terminating DNA replication.

Once replication ceases, DNA ligase forms the final covalent bond between the 5’ and 3’ ends of each daughter DNA strand, resulting in two double-stranded circular E. coli chromosomes.  These chromosomes can then be distributed to daughter E. coli cells after cell division.

Occasionally, the two chromosomes produced by DNA replication are intertwined like the links in a chain. These intertwined DNA molecules are called catenanes. Catenanes must be separated prior to the division of the E. coli cytoplasm, so that each daughter cell receives a chromosome. DNA gyrase solves this catenane problem by cutting one chromosome (both DNA strands are cut), passing the other chromosome through the break, and sealing the break to generate two separate chromosomes that can be distributed properly to the daughter bacterial cells.

EDI_6.9_Termination_of_Replication_in_E._coli-01-01_1.jpgFigure 6.9 Termination of Replication in E. coli --- Image created by KMD

Key Questions

B. DNA Replication in Eukaryotes

Eukaryotic Origins

Eukaryotic DNA replication is more complex than DNA replication in bacteria. This increase in complexity is because eukaryotic genomes are generally larger than prokaryotic genomes, and the genetic material in eukaryotes is organized into linear chromosomes. However, the good news is that the DNA replication process is similar in prokaryotes and eukaryotes and many of the DNA replication proteins (helicases, primases, and polymerases) identified in bacteria have eukaryotic counterparts that function in the same way.  In contrast, one major difference between prokaryotic and eukaryotic DNA replication is that eukaryotic chromosomes have multiple replication origins (see figure 6.10). Like bacteria, DNA replication proceeds bidirectionally from each origin, with the formation of two replication forks per origin. As DNA replication occurs, the replication forks from adjacent origins fuse, eventually producing two identical sister chromatids.

In a model eukaryotic organism, the bread yeast Saccharomyces cerevisiae, the 250–400 origins are called ARS elements. S. cerevisiae ARS elements have the following features:

The DNA replicated from a single ARS element is called a replicon. Since eukaryotic organisms have many origins, eukaryotes also have many replicons.  For example, S. cerevisiae contains 250–400 replicons per genome, while the human genome is thought to contain approximately 25,000 replicons.

6.10_Eukaryotic_Chromosomes_Have_Multiple_Origins.jpgFigure 6.10 Eukaryotic Chromosomes Have Multiple Origins --- This image is used  from OpenStax (access for free at https://openstax.org/books/biology-2e/pages/1-introduction)

Key Questions

Replication Initiation in Eukaryotes

A multi-subunit prereplication complex (preRC) assembles on each ARS element and initiates DNA replication in eukaryotes (see figure 6.11). The preRC contains the following protein components:

After the DNA strands have separated, replication protein A (RPA) prevents the separated DNA strands from reforming hydrogen bonds. The eukaryotic DNA polymerases can then begin the elongation stage of DNA replication.

EDI_6.11_Replication_Initiation_in_Eukaryotes-01-01_1.jpgFigure 6.11 Replication Initiation in Eukaryotes --- Image created by SL

Key Questions

Replication Elongation in Eukaryotes

MCM helicase continues DNA strand separation during the elongation phase of DNA replication, causing the replication forks to proceed in both directions away from each origin. RPA prevents the separated DNA strands from reforming hydrogen bonds.  The separation of the DNA strands by MCM helicase generates positive supercoiling ahead of each replication fork. Topoisomerase II is located ahead of each replication fork and produces negative supercoiling to compensate for the positive supercoiling produced by MCM helicase. Topoisomerase II cleaves ATP to generate negative supercoils.

There are over a dozen different DNA polymerases in a typical eukaryotic cell. These eukaryotic DNA polymerases are named according to the Greek alphabet (α, β, γ, etc.). DNA polymerases alpha (α), delta (δ), and epsilon (ε) are the DNA polymerases involved in replicating nuclear DNA in eukaryotes (see figure 6.12). DNA polymerase α binds to DNA primase to form a protein complex that synthesizes hybrid nucleic acid strands composed of 10 RNA nucleotides followed by 10–30 DNA nucleotides.  These hybrid nucleic acid strands are used as primers by DNA polymerases δ and ε. DNA primase synthesizes the RNA component of the hybrid primer, while DNA polymerase α synthesizes the DNA component of the hybrid primer. Note that DNA polymerase α has both 5’ to 3’ polymerase and 3’ to 5’ exonuclease (proofreading) activity. Once the primer is made, DNA polymerase α is released and is replaced by either DNA polymerase δ or DNA polymerase ε (i.e., the so-called polymerase switch).

DNA polymerases δ and ε are the processive eukaryotic DNA polymerases. These two DNA polymerases bind to proliferating cell nuclear antigen (PCNA), a protein that functions as a sliding clamp, increasing the processivity of DNA polymerases δ and ε. Once bound to PCNA, DNA polymerase ε synthesizes the leading strand, whereas the PCNA:DNA polymerase δ complex synthesizes the lagging DNA strand. Both DNA polymerases ε and δ contain 5’ to 3’ polymerase and 3’ to 5’ exonuclease (proofreading) activity. All three eukaryotic DNA polymerases (α, δ, and ε)  cleave dNTPs during DNA synthesis.  The released energy powers DNA replication, while the nucleoside monophosphates (dAMP, dTMP, dCMP, and dGMP) are incorporated into the growing daughter DNA strands.

Finally, flap endonuclease (Fen1) removes the RNA nucleotides of each primer, and DNA ligase I forms the final covalent bonds to link adjacent Okazaki fragments in the lagging DNA strands. DNA ligase I cleaves ATP during ligation.

6.12_Replication_Elongation_in_Eukaryotes.jpgFigure 6.12 Replication Elongation in Eukaryotes --- Image created by SL.

Key Questions

Replication at Chromosome Ends

The 3’ ends of the parental DNA strands within linear eukaryotic chromosomes present a potential problem during DNA replication. Suppose a primer is made for the daughter DNA strand directly opposite the 3’ end of the parental DNA strand. Once this primer is used for DNA synthesis, the primer is removed with the hope that DNA replication will fill in the primer gap. However, DNA polymerases cannot fill in the primer gap at the end of the chromosome because DNA polymerases require a 3’-OH group to begin DNA synthesis. As a result, this primer gap is not filled in, and the newly synthesized daughter DNA strand is slightly shorter than its template DNA strand. This end replication problem would result in the progressive shortening of daughter DNA strands with each round of DNA replication. Eventually, this shortening would delete genes and have a negative effect on the phenotype of the cell.

Eukaryotes solve this potential DNA replication problem by using telomerase to add moderately repetitive DNA sequences to the 3’ ends of the parental DNA strands prior to DNA replication (see figure 6.13). Telomerase is an unusual enzyme that contains a built-in RNA component (TERC) and a protein component (TERT).  Thus, telomerase is an example of a ribonucleoprotein. The TERC component forms hydrogen bonds with the 3’ overhang DNA sequence at the ends of the two parental DNA strands. Once bound to the 3’ end of the parental DNA strands, TERT catalyzes the synthesis of additional telomere repeat sequences using the built-in TERC component of telomerase as a template. The synthesis of additional telomere repeats by telomerase occurs in the 5’ to 3’ direction. Because telomerase synthesizes DNA in the 5' to 3' direction and requires a 3'-OH group for DNA synthesis, telomerase is considered a DNA polymerase.

Once the 3’ end of the parental DNA strand is lengthened by telomerase, DNA replication of the daughter DNA strand can occur by the synthesis of a primer opposite the repeats added by telomerase. DNA synthesis from this newly added primer occurs using the DNA polymerase δ.  Finally, the primer is removed by Fen1.  Since the primer for the daughter DNA strand is made opposite the telomere repeat sequences added by telomerase, the loss of the primer does not affect structural genes or the phenotype of the daughter cell. 

To sum this all up, telomerase lengthens the parental DNA strands prior to DNA replication, so that the replication enzymes can make the daughter DNA strands shorter. The net result is that the overall chromosome length does not change significantly because of DNA replication.

6.13_Telomerase_Mechanism.jpgFigure 6.13 Telomerase Mechanism --- This image is used  from OpenStax (access for free at https://openstax.org/books/biology-2e/pages/1-introduction)

Key Questions

Review Questions

Fill in the Blank:

  1. The enzyme _________________ methylates adenine to activate DNA replication in bacteria.
  2. The enzyme _______________ connects adjacent Okazaki fragments together during DNA replication in E. coli.
  3. The ______________ protein is the eukaryotic equivalent of SSBPs.
  4. The enzyme _______________ is composed of two subunits, called TERC and TERT.
  5. During DNA replication, the template DNA strands are read by DNA polymerases in the ______________ direction, while the daughter DNA strands are synthesized in the _____________ direction.
  6. Phosphorylation of ____________ and ____________ initiates DNA replication in eukaryotic organisms.
  7. _____________ is a eukaryotic enzyme that produces replication forks, while _____________ is an E. coli enzyme that alleviates positive supercoiling ahead of each replication fork.
  8. The _____________ subunit of the DNA polymerase III holoenzyme is responsible for proofreading, while the _____________ subunit is responsible for DNA synthesis.
  9. _______________ is an unusual DNA polymerase that contains a built-in RNA template molecule.
  10. The enzyme ______________________ has both 5’- 3’ polymerase and 5’ - 3’ exonuclease activity.
  11. ___________ binds directly to the ARS element, while __________________ synthesizes the leading DNA strand in eukaryotes.