What is the function of DNA ligase in elongation phase of DNA replication?

Relaxation of Supercoiling

The action of gyrase during DNA synthesis relieves the strain from positive supercoiling by inducing negative supercoils. If DNA gyrase is blocked by fluoroquinolone antibiotics, bacterial growth is inhibited. An example is ciprofloxacin, which is used to treat urinary tract and other bacterial infections.

12.2 Synthesis of Eukaryotic DNA

The same general principles of bacterial replication apply to eukaryotic replication, although there are differences in detail from the bacterial scheme. In eukaryotes, semi-conservative replication occurs, and as was seen in bacteria, one new strand is made continuously and the other in fragments. Both strands are made simultaneously by a replisome consisting of a helicase plus DNA polymerase holoenzyme. Within the holoenzyme, the sliding clamp loader holds each core enzyme and two sliding clamps. In addition, an RNA primer is required to initiate each new DNA strand.

Three DNA polymerases (α, δ, and ɛ) are involved in eukaryotic chromosome replication. DNA polymerase α (Polα-primase) and two associated small proteins are responsible for initiation of new strands. First, the complex makes an RNA primer. Then, polymerase α elongates the RNA primer by making a short piece of DNA about 20 nucleotides long (the initiator DNA, or “iDNA”). The single-stranded regions of the replication fork are covered by replication protein A (RPA), the eukaryotic equivalent of single-strand binding protein. Together, this whole assembly is known as the replisome (Fig. 10.29).

The functional equivalent to the bacterial sliding clamp loader is called replication factor C (RFC) and binds to the iDNA via other proteins in the pre-LC. RFC then loads the sliding clamp (PCNA protein) plus one of two DNA polymerases onto each strand of DNA. DNA polymerase ɛ is used to make the leading strand, whereas DNA polymerase δ is used for the lagging strand. These two DNA polymerases elongate the two new strands. The sliding clamp of animal cells is a trimer (not a dimer as in bacteria) that forms a ring surrounding the DNA. It was named PCNA, for proliferating cell nuclear antigen, before its role was known. Although two different polymerases are usually found in the replisome, at least in yeast, replication can take place without polymerase ɛ—apparently polymerase δ can substitute if necessary. The various families of DNA polymerase are listed in Box 10.03.

Box 10.03

DNA Polymerase Families

DNA polymerases all perform the same basic function, catalyzing the connection of a 5′-phosphate group from the incoming nucleotide to the 3′–OH group on the ribose or deoxyribose of the upstream nucleotide. Yet, there are so many different variations of structure and other functions that DNA polymerases actually fall into seven different families: (Table 10.02).

Linking of the Okazaki fragments differs significantly between animal and bacterial cells. In animals, there is no equivalent of the dual function DNA Pol I of bacteria. The RNA primers are removed by the exonucleases Fen1 and/or Dna2, and the gaps are filled by the DNA polymerase δ that is already working on the lagging strand. As in bacteria, the nicks are sealed by DNA ligase.

Eukaryotic replisomes are more complex than bacterial replisomes. They contain extra regulatory proteins and multiple different forms of DNA polymerase (α, ɛ, and δ).

12.2 Synthesis of Eukaryotic DNA

The same general principles of bacterial replication apply to eukaryotic replication, although there are differences in detail from the bacterial scheme. In eukaryotes, semi-conservative replication occurs, and as was seen in bacteria, one new strand is made continuously and the other in fragments. Both strands are made simultaneously by a replisome consisting of a helicase plus DNA polymerase holoenzyme. Within the holoenzyme, the sliding clamp loader holds each core enzyme and two sliding clamps. In addition, an RNA primer is required to initiate each new DNA strand.

In animal cells, the double helix first needs to be separated into two single strands at the origin of replication. The pre-loading complex (see Fig.10.27) recruits the helicase, minichromosome maintenance (MCM), which then moves along the helix in a 3′ to 5′ direction, separating the two strands of DNA (Fig. 10.28). MCM is similar to the bacterial helicase DnaB, since both molecules consist of multiple subunits. Three DNA polymerases (α, δ, and ɛ) are involved in eukaryotic chromosome replication. DNA polymerase α (Polα-primase) and two associated small proteins are responsible for initiation of new strands. First, the complex makes an RNA primer. Then, polymerase α elongates the RNA primer by making a short piece of DNA about 20 nucleotides long (the initiator DNA, or “iDNA”). The single-stranded regions of the replication fork are covered by replication protein A (RPA), the eukaryotic equivalent of single-strand binding protein.

The bacterial functional equivalent to the sliding clamp loader is called replication factor C (RFC) and binds to the iDNA via other proteins in the complex and loads a sliding clamp (PCNA protein) plus one of two DNA polymerases onto each strand of DNA. DNA polymerase ɛ is loaded onto the DNA for the leading strand, whereas DNA polymerase δ is used for the lagging strand. These two DNA polymerase assemblies elongate the two new strands. The sliding clamp of animal cells is a trimer (not a dimer as in bacteria) that forms a ring surrounding the DNA. It was named PCNA, for proliferating cell nuclear antigen, before its role was fully known. Although two different polymerases are usually found in the replisome, at least in yeast, replication can take place without polymerase ɛ—apparently polymerase δ can substitute if necessary. Eukaryotic replisomes also contain other regulatory proteins such as Cdc45 and a complex of four proteins called GINS for the four protein names, Go, Ichi, Nii, and San. The function of these within the replisome is still under investigation.

Linking of the Okazaki fragments differs significantly between animal and bacterial cells. In animals, there is no equivalent of the dual function DNA polymerase I of bacteria. The RNA primers are removed by the exonucleases Fen1 and/or Dna2, and the gaps are filled by the DNA polymerase δ that is already working on the lagging strand. As in bacteria, the nicks are sealed by DNA ligase.

Eukaryotic replisomes are more complex than bacterial replisomes. For example, eukaryotic replisomes have more regulatory proteins (cdc45 and GINS), and the replisome has different forms of DNA polymerase (α, ɛ, and δ) to synthesize the new DNA.

The Sequence of Primer RNA

In Escherichia coli, large amounts of Okazaki fragments can be isolated from double mutants carrying temperature-sensitive lesions in RNase H and the 5′-exonuclease domain of polymerase I. When these cells are arrested shortly after the initiation of replication, the primer RNA attached to the Okazaki fragments is found to be 11±1 nucleotides long. The primer was found to be initiated with ATP fives times more often than with other nucleoside triphosphates. A phosphodiester bond is formed between this initiating nucleotide and either ATP or GTP to make the initial diribonucleotide. In E. coli, primer synthesis begins complementary to the template trinucleotide sequence 5′-d(CTG)-3′. The guanine in the trinucleotide does not serve as a template for synthesis but is required for directing primase to the cytosine and thymine so that it will synthesize pppApG.

Primase and transcriptional RNA polymerase exhibit a high level of initiation specificity (Table 1). In all cases, once the specific purine-rich diribonucleotide has been synthesized, the rest of the primer sequence is determined by whatever the template sequence happens to be. There are several exceptions to this general rule; P4 primase recognizes only a dinucleotide template sequence; T7 primase synthesizes primarily trinucleotides and tetranucleotides ribopolymers with distinct template sequence specificity (not shown); and second of the two Thermoanaerobic tengcongensis primases completely lacks initiation specificity. This latter case is interesting because sequencing efforts have revealed a number of bacterial genomes that code for two primases, where the second one is less sequence conserved. The role of these additional primases is unclear.

Table 1. Initiation specificity of selected primases and RNA polymerases as established in biochemical assaysa

EnzymeFirst diribonucleotideInitiation trinucleotideBacteriophageP4 α proteinpppApGd(CTN)SP6 gene 4 proteinpppGpCd(GCA)T3 and T7 gene 4 proteinpppApCd(GTC)T4 gene 61 proteinppp(A/G)pCd(GTT) and d(GCT)
BacteriaE. coli RNA polymerasepppApUd(ATG)FirmicutespppAp(A/G)d(CTA) and d(TTA)ProteobacteriapppApGd(CTG) and d(CTA)Aquifex aeolicuspppGpGd(CCC)T. teng. DnaGpppGpGd(CCC)T. teng. DnaG2pppNpNd(NNN)
Archaeal/EukaryoticEukaryoticppp(A/G)p(A/G)d(PyPyC)Herpesviralppp(A/G)p(A/G)d(PyPyG)

The biochemical features of primer RNA synthesis have provided a number of insights into the control of DNA replication. For bacterial and eukaryotic primases, the rate-determining step is either the rate of formation of the first phosphodiester bond or some step preceding it. Rate-limiting steps are usually subject to control. In bacteria, DnaB helicase is able to stimulate primase activity greatly and, because it unwinds duplex DNA, results in the synthesis of primers at the DNA replication fork.

In eukaryotes, replication protein A is a single-stranded DNA binding protein that is able to stimulate eukaryotic primase. After catalyzing the formation of the first bond, primases synthesize the next 10 or so bonds rapidly but then slow down. During this brief elongation phase, bacterial and eukaryotic primases readily incorporate deoxyribonucleotides into the primer to create mixed ribo- and deoxyribo-oligomers. In the absence of other replication enzymes, bacterial primer RNA is 12 or more nucleotides and eukaryotic primer RNA is 8 or more nucleotides. When either DNA polymerases or replicative helicases and their substrates are added to the primase assay mixture, the primers are limited to a length of 7–12 nucleotides. These primer lengths are the same as those observed at the ends of Okazaki fragments isolated from living organisms.

Leading and Lagging Strands

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The lagging strand synthesis is done discontinuously. Okazaki fragments are initiated by creation of a new RNA primer by the primosome. To restart DNA synthesis, the DNA clamp loader releases the lagging strand from the sliding clamp, and then reattaches the clamp at the new RNA primer. Then DNA polymerase III can synthesize the segment of DNA.

One strand of the DNA double helix is easy to replicate because DNA polymerase III can continuously slide along this strand moving towards the fork, all the while inserting complementary bases 5´ to 3´. This strand is called the leading strand. However, on the opposite side, DNA polymerase is still required to synthesize DNA in a 5´ to 3´ direction, but the movement of the replisome relative to the template strand is 5´ to 3´. This strand is called the lagging strand. Since the newly synthesized strand must still be antiparallel to the template, the lagging strand template DNA is looped out away from the replisome and replicated in sections. The sliding clamp loader attaches the sliding clamp to the RNA primer to begin synthesis of the fragments. At the end of each fragment, the sliding clamp releases the DNA and is loaded onto the next RNA primer. Each section begins with an RNA primer. This discontinuous synthesis results in the generation of fragments on the lagging strand called Okazaki fragments.

DNA polymerase I recognizes a “nick” or break in the phosphate backbone, and then removes each RNA primer and fills the gaps with DNA. DNA ligase then covalently links the phosphate backbone.

After the replisome has passed, the lagging strand contains intervening RNA primer sequences along with gaps (missing nucleotides) in the strand and nicks (missing the covalent bond between adjacent nucleotides). More enzymes are needed to clean up the DNA. DNA polymerase I removes the RNA primer and fills in the gaps with DNA. However, DNA polymerase I cannot catalyze the reaction to remove the nicks. Another enzyme, DNA ligase, seals the nicks by forming the phosphodiester bond, thus generating a continuous sugar-phosphate backbone for the lagging strand.

2.3.2.3 DNA Ligases

DNA ligases are best known for their role in joining adjacent Okazaki fragments at the lagging strand of the replication fork; however, they are essentially involved in any process that requires sealing of phosphodiester bonds from the DNA backbone. Ligases catalyze the formation of phosphodiester bond between 3′OH and 5′phosphate on various substrates such as DNA nicks, DNA fragments with various lengths cohesive ends, DNA fragments with blunt ends and some DNA/RNA hybrids. Fig. 2.14 depicts the basics of ligase action at a DNA nicked substrate. The most widely used viral ligase is the T4 DNA ligase, which is routinely employed in cloning applications. T4 DNA ligase exhibits highest activity ligating DNA nicks and fragments with overhangs longer than 2 nucleotides, whereas fragments with blunt ends, shorter overhangs or nicks containing mismatches are processed less efficiently requiring higher enzyme concentrations and extended ligation times. Interestingly, T4 ligase can also join the 3′ end of RNA strand to a DNA strand with 5′ phosphate when the DNA fragment is annealed to a complementary DNA. Many commercial formulations of T4 ligase are available, which differ in enzyme concentration, presence of proprietary ligase activity enhancers, or buffer conditions compatible with various downstream applications such as transformation of chemical or electrocompetent cells. DNA ligases from bacteriophages T3 and T7 are also commercially available, as well as several nonviral DNA ligases, some of which are thermostable. T7 DNA ligase is the enzyme of choice when sticky ends only need to be sealed since its affinity to sticky ends grossly outweighs the one for blunt ends. T3 ligase is more salt tolerant than T4 and T7 ligases and it is the preferred ligase in applications requiring high ionic strength conditions. The use of DNA ligases in cloning is on decline due to the development of ligation-independent cloning approaches (Fig. 2.9) and recombination-based cloning technologies (Chapter 3), however, their overall value as molecular biology reagents is increasing as more and more techniques employing adapter ligation (for example, SAGE, Fig. 2.7) are being developed. Some of the new-generation sequencing methods also employ T4 DNA ligase to attach adapters to genomic DNA fragments to be sequenced.

Ligase itself drives sequencing by ligation, the newest concept in the fast changing landscape of DNA sequencing. Currently, two major platforms for whole genome sequencing employ the principle: SOLiD (Thermo Fisher, Inc.) and Complete Genomics (Beijing Genomics Institute). The SOLiD technology immobilizes DNA fragments subject to sequencing on beads attached to a slide. Bead/DNA attachment is accomplished via universal adapter. An anchor sequence, complementary to the adapter provides the 5’ phosphate group to be ligated to the 3’ hydroxyl group of the upcoming probe. Fluorescent sequencing probes consist of two known nucleotides in position 1 and 2 (depicted in color in Fig. 2.15), followed by a stretch of degenerate or universal bases depicted with N and Z. At each cycle only the probe with a perfect match of the two known nucleotides will ligate. The slide is imaged and the unextended molecules are capped by unlabeled probes to maintain cycle synchronization. The fluorophore and the terminal degenerated bases are cleaved off the probe, resulting in net 5 nt extension. The process is repeated 10 times resulting in a string of nucleotides in which two out of every five bases are identified. All ligated probes are then removed, and the entire process of probe binding, ligation, imaging, and cleavage is repeated four times, each with different anchors. At each round the new anchor is offset with one nucleotide allowing the entire sequence to be deciphered. The sequence is assembled computationally based on the imaging snapshots taken after each cycle.

The Complete Genomics technology employs fluorescent probes containing only one known nucleotide and several degenerate nucleotides. The DNA genome of interest is fragmented, cloned with flanking synthetic adapters, and then amplified to form DNA nanoballs. Each DNA nanoball is a long concatemer, product of rolling circle DNA amplification with bacteriophage phi29 DNA pol. Arrays of nanoballs are assembled in a flow cell, which is imaged after every sequencing cycle. Each cycle starts with the hybridization of an anchor sequence to one of the synthetic adapters. Then a pool of fluorescently labeled probes is flown allowing complementary probe hybridization and ligation to take place. The pool of unligated probes is washed away, and the flow cell is imaged. The same steps are performed repeatedly using a set of anchors with offset of one nucleotide, and the DNA sequence is assembled computationally. A separate sequence-specific set of anchors is used for each adapter. Sequences derived from different library clones are assembled into the sequence of the entire genome.

Cellular Functions of Eukaryotic DNA Ligases

As DNA joining is required to complete DNA replication, DNA repair, and genetic recombination, it appears likely that the multiple species of DNA ligase have evolved to participate in specific DNA transactions. Insights into the cellular functions of the different DNA ligases have been obtained by various approaches including phenotypic analysis of DNA ligase-deficient cells and the identification of interacting proteins (Table 1). The results of these studies are summarized below.