What is the role of DNA ligase in the elongation of the lagging strand during DNA replication A It synthesizes RNA nucleotides to make a primer?

Primer RNA

M.A. Griep, in Encyclopedia of Genetics, 2001

Definition

Primer RNA is RNA that initiates DNA synthesis. Primers are required for DNA synthesis because no known DNA polymerase is able to initiate polynucleotide synthesis. DNA polymerases are specialized for elongating polynucleotide chains from their available 3′-hydroxyl termini. In contrast, RNA polymerases can elongate and initiate polynucleotides. Primases are special RNA polymerases that synthesize short-lived oligonucleotides used only during DNA replication.

Even though ‘transcriptional’ RNA polymerases primarily synthesize messenger RNA, transcripts are sometimes used to initiate DNA synthesis. For instance, the single-stranded DNA phage M13 genome utilizes RNA polymerase instead of primase to initiate its DNA synthesis. In addition, the dominant hypothesis concerning mitochondrial DNA replication initiation is that the mitochondrial RNA polymerase synthesizes a polymer that is not displaced from the template. Then, the special RNase MRP cleaves the ribopolymer at specific sites enabling the exposed 3′-hydroxyl termini to serve as primers for DNA synthesis. Finally, transfer RNAs make up a special class of primer RNA because certain species of tRNA are used by retroviral reverse transcriptases to initiate replication of retroviral genomes. It is also possible to initiate DNA synthesis without primer RNA. The initiator proteins of adenovirus and ϕ29 covalently attach to both of the 5′-ends of linear duplex DNA and provide a serine β-hydroxy group from which a DNA polymerase elongates. Another example is that many plasmids encode sequence-specific nucleases which cleave one strand of the duplex to create a 3′-hydroxyl for the host DNA polymerase. An example of an animal virus is parvovirus, where the 3′-end of the parental strand forms a DNA hairpin and becomes the primer of its complement.

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Primer RNA☆

M.A. Griep, in Reference Module in Life Sciences, 2017

Abstract

Primer RNA is RNA that initiates DNA synthesis. Primers are required for DNA synthesis because no known DNA polymerase is able to initiate polynucleotide synthesis. Edited transcriptional RNA is used to initiate DNA synthesis in some phage and in metazoan mitochondria. Transfer RNAs are used by retroviral reverse transcriptases to initiate replication of retroviral genomes. Bacteria, archaea, and eukaryotes use an enzyme called primase to synthesize short-lived oligonucleotides used only during DNA replication. These primases synthesize a primer RNA once on each leading strand template to initiation DNA synthesis and repeatedly on the lagging strand template to initiate Okazaki fragment synthesis. Primase initiate synthesis from specific trinucleotides that differ according to the organism’s phylogeny. Once the specific purine-rich diribonucleotide has been synthesized complementary to the 5′ and central nucleotides of the initiation trinucleotide, the rest of the primer sequence is synthesized in a template-dependent manner. In bacteria, the process of removing the primers is not directly coupled to discontinuous synthesis and involves such enzymes as RNase H and the 5′-exonuclease of DNA polymerase I.

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Quantum Leaps in Biochemistry

Anil Day, Joanna Poulton, in Foundations of Modern Biochemistry, 1996

Mitochondria Import RNA

The discovery that the RNA primer used to initiate DNA synthesis in mammalian mitochondria was imported from the nucleus dispelled the belief that only proteins but not RNA could cross organelle membranes (Chang and Clayton, 1987). Evidence that tRNAs are imported from the cytosol into mitochondria is derived from elucidating the coding capacity of mitochondrial DNA and also characterizing tRNAs present in isolated mitochondria. Mammalian mitochondria use 22 unusual tRNAs to decode the 61 sense codons. The linear 15.8-kb genome of C. reinhardtii only encodes three tRNAs (Michaelis et al., 1990). Although liverwort mitochondrial DNA encodes 27 tRNA species, two species necessary to read leucine and threonine codons are absent. C. reinhardtii, plant, and trypanosome mitochondria appear to import nuclear-encoded tRNAs to make up a complete set for protein synthesis. Eleven of the 31 tRNA species present in potato mitochondria are encoded by nuclear DNA and are imported from the cytosol (Dietrich et al., 1992). The plastid genome of Epifagus virginiana, a nonphotosynthetic parasite of beech trees, lacks 13 tRNA genes found in green plastids. This suggests tRNA import can also occur in plastids (Wolfe et al., 1992).

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Primase☆

M.A. Griep, J. Periago, in Reference Module in Life Sciences, 2017

Definition

Primase is the enzyme that synthesizes RNA primers. Primers are oligonucleotides that are complementarily bound to a DNA template and from which DNA polymerases elongate. Special proteins are responsible for loading primase at the origin of replication so that leading strand DNA synthesis can commence. In a subsequent step, other replication proteins cause primase to initiate DNA replication on the opposite lagging strand. After both the leading and lagging strand primers have been elongated by DNA polymerases, the RNA primers are enzymatically eliminated and the resulting gap in the DNA sequence is filled in by DNA polymerase I and DNA ligase.

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Primase

M.A. Griep, in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Abstract

Primase is the enzyme that synthesizes RNA primers, oligonucleotides that are complementarily bound to a nucleic acid polymer. Primase is required because DNA polymerases cannot initiate polymer synthesis on single-stranded DNA templates; they can only elongate from the 3′-hydroxyl of a primer. Primases fall into two major sequence and structure families: bacterial and archaeal/eukaryotic nuclear. Bacterial primases are monomers consisting of three domains. The N-terminal domain has a zinc-finger motif and is likely responsible for the initiation specificity of this enzyme. The central catalytic domain binds single-stranded DNA and catalyzes RNA polymer initiation and elongation complementary to it. The C-terminal domain interacts with other proteins, including DnaB helicase so that its activity takes place at the replication fork. The bacterial primase gene, dnaG, is the central gene of the macromolecular synthesis operon carrying the genes for the initiation phases of translation, replication, and transcription. Of the three genes, dnaG is under the most levels of control and is expressed in the lowest amount. Archaeal/eukaryotic primase resides in a heterotetramer consisting of a small primase subunit, a large primase subunit, a regulatory phosphoprotein, and DNA polymerase alpha. The small subunit has primer synthesis activity that is modulated by the other three proteins in the complex as well as by Replication protein A, a single-stranded DNA-binding protein required for lagging strand DNA synthesis, and the GINS complex, the central hub around which the leading- and lagging-strand DNA replicases assemble to control the progression of the replication fork. GINS interacts with the MCM helicase that translocates on the leading-strand template and also interacts with the DNA polymerase alpha/primase complex on the lagging strand.

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DNA Replication Fork, Bacterial

M.M. Spiering, S.J. Benkovic, in Encyclopedia of Biological Chemistry (Second Edition), 2013

Lagging-Strand Synthesis

Synthesis of the lagging strand at the replication fork begins with an RNA primer that is made by a primase. However, because the lagging strand is made in short Okazaki fragments, several RNA primers are necessary in a repeating cycle of primer initiation, elongation by DNA polymerase, and ligation to seal the fragments (Figure 2). Okazaki fragments are 1000–3000 bases in length and replication is proceeding at 400–1000 bases s−1, so the cycle of reactions on the lagging strand must be completed in a few seconds. Reactions required to prime the next fragment, elongate the present fragment, and remove primers from the previous fragments can occur simultaneously allowing the lagging strand cycle to be completed rapidly.

Beginning in the elongation stage of this cycle, the lagging-strand polymerase holoenzyme is synthesizing an Okazaki fragment that began with the primer labeled P2 (Figure 2(a)). At the same time, the helicase surrounding the lagging-strand template at the fork is unwinding the duplex ahead of the leading-strand polymerase holoenzyme. The ssDNA being generated by the helicase along with the ssDNA remaining to be replicated ahead of the lagging-strand polymerase holoenzyme is coated with SSB proteins.

As the lagging strand loop continues to grow due to the activities of the helicase and lagging-strand polymerase holoenzyme, the 5′–3′ nuclease is removing the RNA primer (P1) and a small amount of adjacent DNA from the previous fragment (Figure 2(b)). The removal of this DNA first added to the primer may increase the accuracy of replication.

When the lagging-strand polymerase holoenzyme completes the nascent fragment, creating a nick, the clamp releases the polymerase and stays behind on the DNA (Figure 2(c)). In E. coli, this release of the polymerase core from the β-clamp is facilitated by the C-terminal domain of the τ-subunit of the clamp loader acting as an unloader. There is no evidence that a clamp loader is needed to disengage the clamp and polymerase in the T4 system. If the polymerase fails to be released at the nick, it can continue to synthesize DNA displacing the 5′ end of the downstream fragment forming a flap. However, all of the prokaryotic 5′ nucleases involved in primer removal also have flap endonuclease activities able to remove these displaced strands. The nicks that are formed between adjacent fragments are sealed by DNA ligase.

When the synthesis of the nascent fragment is complete, primase, associated with the helicase at the fork, makes the RNA primer (P3) that will be used to start the next fragment (Figure 2(c)). A new clamp is loaded on to the primer in the E. coli and T4 systems by the clamp loader. The E. coli clamp loader is bound to the two polymerases as well as the helicase at the fork, while the clamp is recruited from solution. Dilution experiments suggest that both the T4 clamp and clamp loader must be provided from solution for each cycle of lagging strand synthesis. Sometimes, the primase makes a primer before the lagging-strand polymerase holoenzyme finishes the previous Okazaki fragment. This acts as a signal that causes the lagging-strand polymerase to be released prematurely leaving a gap between fragments that must ultimately be repaired by loading another polymerase.

Finally, the lagging-strand polymerase is transferred to the clamp-loaded new primer (P3) to begin synthesis of the next fragment (Figure 2(d)). In the case of T7, the DNA-binding domain of the primase binds the new primer and transfers it directly to the lagging-strand polymerase. The same lagging-strand polymerase is recycled from one Okazaki fragment to the next being held at the replication fork by interactions with the clamp loader in E. coli, the leading-strand polymerase in T4, or the helicase in T7.

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Nucleases

Hyone-Myong Eun, in Enzymology Primer for Recombinant DNA Technology, 1996

ii. Physiological roles of RNase H.

Despite multiple roles proposed for RNase H, for example, the removal of RNA primers from Okazaki fragments during DNA replication, no in vivo roles have been clearly established in eukaryotes (56). In fact, the physical differences between eukaryotic and prokaryotic RNases H and among various RNases H subspecies from a single eukaryotic cell type suggest that their in vivo roles may be just as diverse.

Available evidence suggests that RNases HI in E. coli may not be indispensable for cell survival (57). A RNase H mutant, generated by ethylmethane sulfonate mutagenesis, exhibits less than 8% of the wild type activity but has no apparent phenotypic differences (53). A 15-fold overexpression of the cloned rnh gene has no detectable effect on cell growth (42). Although RNase HI-deficient mutants generally exhibit no grossly altered phenotype, they display some abnormalities such as (a) sustained DNA replication in the absence of protein synthesis, (b) lack of the requirement for dnaA protein and the origin of replication (oriC), and (c) growth sensitivity to rich media (58). The RNase H of E. coli is presumed to play a role in (i) the replication of chromosomal DNA, (ii) the replication of plasmid (ColEl) DNA at the precise replication origin (59, 60) and functioning as a “renaturase” factor, and (iii) the transcript displacement by RNA polymerase (61). Perhaps some of these in vivo functions are substituted, when necessary, by other E. coli enzymes that are known to exhibit RNase H-type activity, albeit with less specificity and efficiency. The possibility of RNase H activity substitution has become more realistic in light of the discovery of a second RNase H gene in E. coli. In addition, DNA Pol I and exonuclease III can degrade either the DNA or RNA of the hybrids.

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Cell Division and DNA Replication

David P. Clark, Nanette J. Pazdernik, in Molecular Biology (Second Edition), 2013

Leading and Lagging Strands

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.

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DNA Replication Fork, Eukaryotic

L.M. Kelman, Z. Kelman, in Encyclopedia of Biological Chemistry (Second Edition), 2013

DNA Polymerase α/Primase Complex

DNA polymerases are incapable of initiating DNA synthesis de novo and require DNA primases, which synthesize short RNA primers on template DNA that are subsequently extended by the polymerase. In eukarya, DNA primase is part of a four-subunit complex, the Polα/primase complex, containing subunits with molecular masses of 180, 68, 58, and 48 kDa. Two of the subunits, p48 and p58, are required for primase activity, with p48 serving as the catalytic unit. Primase synthesizes short RNA primers (8–12 nucleotides (nt)), which are then elongated by polymerase α (Polα) to ~30 nt, forming pre-Okazaki fragments. These RNA–DNA hybrid molecules are recognized by the polymerase accessory complex, replication factor C (RFC), to initiate processive DNA synthesis by the replicative polymerase. On the lagging strand, Polα/primase activity is required at the initiation of each Okazaki fragment.

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Inoviruses

L.A. Day, in Encyclopedia of Virology (Third Edition), 2008

DNA replication and gene expression

The ssDNA is converted to a parental supercoiled dsDNA by cellular enzymes starting with RNA polymerase which creates an RNA primer for the initial complementary strand synthesis. The DNA site for the priming has an unusual hairpin structure with a high affinity for the RNA pol holoenzyme. Priming is followed by complementary strand synthesis by a DNA polymerase, then displacement of the RNA primer and ligation, and finally gyrase action, to produce the parental supercoiled dsDNA. Transcripts are generated from promoters of various strengths and various rho-dependent and -independent terminators. Translation is controlled by overlaps of genes in different frames or alternate starts in the same frame as well as by codon usage. Replication of this dsDNA begins with a nick at a specific, high-symmetry site once a viral endonuclease is expressed from it. Progeny dsDNA circles are produced via ssDNA intermediates in rolling circle replication. The dsDNA circles lead to further mRNA and protein synthesis. Increasing amounts of another replication protein and a ssDNA-binding protein eventually downregulate the nicking activity of the nuclease. Large amounts of the DNA binding protein (gene 5 protein) accumulate to form complexes with nascent plus strands being displaced in the rolling circle replication. About 100 progeny ssDNA plus strands become sequestered within flexible unbranched filamentous structures having stoichiometric ratios of 9 or 10 nt per gp5 dimer. The crystal structure of gp5 has been determined and the structures and properties of gp5/nucleic acid complexes have been extensively studied. Nevertheless, the complex mechanism of gp5 slippage on the lengthening DNA to form such structures, which involves the gp5 folding the nascent single strand back on itself and continually adjusting the fold point, is not established. The mechanism leaves the tight hairpin of the packaging site hairpin exposed at one end and the rest of the loop of ssDNA covered with about 700 gp5 dimers. While this is happening, thousands of copies of the major coat protein and smaller numbers of the four other capsid proteins are accumulating in the inner membrane. The morphogenesis proteins are also inserted in the inner membrane, and about 14 molecules of another morphogenetic protein forms a closed pore in the outer membrane.

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What is the role of DNA ligase in the lagging strand?

On the lagging strand, DNA synthesis restarts many times as the helix unwinds, resulting in many short fragments called “Okazaki fragments.” DNA ligase joins the Okazaki fragments together into a single DNA molecule.

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

In the final stage of DNA replication, the enyzme ligase joins the sugar-phosphate backbones at each nick site. After ligase has connected all nicks, the new strand is one long continuous DNA strand, and the daughter DNA molecule is complete.

What is the function of DNA ligase quizlet?

DNA ligase joins pieces of DNA together, mainly joins Okazaki fragments with the main DNA piece.

What is the role of the DNA ligase in DNA replication apex?

DNA Ligase enzymes seal the breaks in the backbone of DNA that are caused during DNA replication, DNA damage, or during the DNA repair process.