NUCLEIC ACID SYNTHESES

A. Similarity of DNA and RNA Synthesis

All nucleic acids are usually synthesized by DNA template-guided polymerization of nucleotides—ribonucleotides for RNA and deoxy(ribo)nucleotides for DNA. The reactant monomers are 5′ ribonucleoside (or deoxyri-bonucleoside) triphosphates. These can be described in the following chemical equations:

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and

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Enzymatic polymerization is carried out by DNA and RNA polymerases, both of which carry out pyrophos-phorolysis, i.e., cleavage of a high energy pyrophosphate bond coupled to esterification of 5′ phosphate linked to the 3′-OH of the previous residue. The reaction is reversible, although it strongly favors synthesis. Degradation of nucleic acids is not due to reversal of the reaction, but rather a hydrolytic reaction catalyzed by nucleases, namely, RNases and DNases, which generate nucleotides or deoxynucleotides, respectively.


Three distinct stages are involved in the biosynthesis of both DNA and RNA: initiation, chain elongation, and termination. Initiation denotes de novo synthesis of a nucleic acid polymer which is generally well regulated by complex processes, as described later. The key difference in initiation of a DNA vs RNA chain is that RNA polymerases can start a new chain, while all DNA poly-merases require a "primer," usually a short RNA or DNA sequence with a 3′-OH terminus, to which the first de-oxynucleotide residue is added. Elongation denotes continuing polymerization of the monomeric nucleotides, and termination defines stoppage of nucleotide addition to the growing polymer chain.

During synthesis the enzymes catalyzing the polymerization reaction are guided by nucleic acid templates that provide the complementary sequence for the incorporated nucleotides (Fig. 4). The basic catalytic enzyme in such reactions is called DNA or RNA polymerase. In cells the template for both DNA and RNA is genomic DNA. There are some exceptions to these general rules. Some DNA polymerases can synthesize homo- or heteropolymers of deoxynucleotides in vitro in the absence of a template; the substrate is restricted to one or two dNTPs. While it is unlikely that these homo- or heteropolymers, e.g., (dA^dT)n or poly(dA)n^poly(dT)n, are formed in vivo, the availability of these polymers significantly advanced our understanding of the properties of DNA, before the age of chemical or enzymatic oligonucleotide synthesis.

There are some exceptions to the norm of DNA-dependent DNA or RNA synthesis, mostly in lower eukaryotes or viruses (Fig. 5). One example is RNA-dependent RNA synthesis in plant, animal, or bacterial viruses. In these cases, a single-stranded RNA template rather than double-stranded DNA guides synthesis of the complementary RNA strand, based on conventional base pairing. The polarity of RNA adds a level of complexity during synthesis. Thus, the RNA genome of a virus that can be directly read and thus provides the mRNA function is called the positive strand, as in polio virus. In this case, the viral genome RNA functions as the mRNA and encodes the RNA polymerase, which is synthesized like other viral proteins in the infected cell. This RNA polymerase subsequently synthesizes the complementary negative strand, which then serves as the template for synthesis of the progeny positive strand RNA. The progeny RNA is then packaged into mature progeny virus.

Replication of circular DNA of prokaryotes and viruses, plasmids, and mitochondria. The basic steps of replication are shown. (A) Rolling circle mode of replication for single-stranded circular DNA: single-stranded (ss) DNA is replicated to the replicative form (RF), which then acts as the template for progeny ssDNA synthesis via a rolling circle intermediate. (B) Circular duplex DNA can be replicated at the orisite by formation of a 6 intermediate. Replication could be bidirectional (as shown here) or unidirectional. 5' ^ 3' chain growth dictates that DNA synthesis is continuous on one side of the ori and discontinuous on the other side for each strand; (+) and (-) strands are shown to distinguish the strand types. (C) Replication of a linear genome with multiple origins.

FIGURE 4 Replication of circular DNA of prokaryotes and viruses, plasmids, and mitochondria. The basic steps of replication are shown. (A) Rolling circle mode of replication for single-stranded circular DNA: single-stranded (ss) DNA is replicated to the replicative form (RF), which then acts as the template for progeny ssDNA synthesis via a rolling circle intermediate. (B) Circular duplex DNA can be replicated at the orisite by formation of a 6 intermediate. Replication could be bidirectional (as shown here) or unidirectional. 5′ ^ 3′ chain growth dictates that DNA synthesis is continuous on one side of the ori and discontinuous on the other side for each strand; (+) and (-) strands are shown to distinguish the strand types. (C) Replication of a linear genome with multiple origins.

In contrast, the genomic RNA of negative strand viruses (e.g., vesicular stomatitis virus) cannot function directly as mRNA and thus cannot guide synthesis of proteins, including the RNA replicase, by itself after the infection of host cells. These viruses carry their own RNA replicase within the virion capsids, which carry out (+) mRNA strand synthesis after infection (Fig. 5).

Retroviruses comprise diverse groups of viruses, including human immunodeficiency virus (HIV), which share a common mechanism of genome replication. The RNA genomes of these viruses encode an RNA-dependent DNA polymerase (reverse transcriptase or RT) which first generates the complementary (c) DNA of the viral genome. RT has also RNaseH (specific nuclease for degrading RNA from RNA-DNA hybrids) and DNA-dependent DNA polymerase activities. After copying the RNA template, the enzyme degrades the RNA and is able to convert the resulting single-stranded cDNA to duplex DNA. This is then integrated into the host cell genome as proviral DNA, from which the progeny viral RNA is eventually transcribed. Thus, the reverse transcriptase is an unusual polymerase because it can utilize both RNA and DNA templates (Fig. 5). There is strong evidence that such reverse transcription was involved in synthesis of "retro-transposons," a special class of mobile genetic elements, during the evolution of mammalian genomes. These mobile genetic elements, also known as transposons, when identified in bacteria and lower eukaryotes, consist of specific DNA sequences which can be relocated randomly in the genome. The transposition is mediated by enzymes called transposase, usually synthesized by a gene in the transposon. During transposition ofretransposons, certain mRNAs are reverse transcribed and then integrated into the genome like the proviral sequence. The presence of specific flanking sequences allows these elements to relocate to other sites in the genome.

Replication of mammalian viral RNA genome. The basic steps of replication are shown for (A) a (+) strand genome, which acts as an mRNA for encoding viral proteins; (B) a (-) viral genome cannot encode protein and first has to be replicated by the RNA replicase (•) which is present in the virus particle. Once the complementary (+) strand which serves as mRNA is synthesized, viral-specific proteins are synthesized, including RNA replicase. (C) Replication of (+) stranded retroviral genomes first involves synthesis of the reverse transcriptase which directs synthesis of duplex DNA in two stages from the RNA template. Circularization of the DNA followed by its genomic integration allows synthesis of progeny viral RNA by the host transcription machinery.

FIGURE 5 Replication of mammalian viral RNA genome. The basic steps of replication are shown for (A) a (+) strand genome, which acts as an mRNA for encoding viral proteins; (B) a (-) viral genome cannot encode protein and first has to be replicated by the RNA replicase (•) which is present in the virus particle. Once the complementary (+) strand which serves as mRNA is synthesized, viral-specific proteins are synthesized, including RNA replicase. (C) Replication of (+) stranded retroviral genomes first involves synthesis of the reverse transcriptase which directs synthesis of duplex DNA in two stages from the RNA template. Circularization of the DNA followed by its genomic integration allows synthesis of progeny viral RNA by the host transcription machinery.

B. DNA Replication vs Transcription: Enzymatic Processes

The broad chemical steps in DNA and RNA synthesis are quite similar, in that both processes represent reading of a DNA strand as the template. However, while both strands of DNA have to be copied, transcription is polar because only one strand is normally copied into RNA whose sequence is identical to the other strand (except for replacement of thymidine by uridine). This is achieved by the presence of discrete start and stop signals bracketing "transcription units" corresponding to each gene containing unique sequences, called promoters; their sequences provide the recognition motif for RNA poly-merase to bind and start RNA synthesis unidirectionally. Similarly, the stop sequences are recognition motifs for the transcription machinery to stop and fall off the DNA template.

As mentioned before, the two strands of a DNA double helix are of opposite polarity, i.e., one strand is in the 5′ ^ 3′ orientation and its complementary strand in the 3′ ^ 5′ orientation. Furthermore, the fact that all nucleic acid polymerases can polymerize nucleotide monomers only in the 5′ ^ 3′ direction as guided by base pairing with a template does not pose a problem for RNA synthesis because only the 3′ ^ 5′ strand of the DNA template is copied. However, DNA replication, where both strands have to be copied in the same 5′ ^ 3′ direction of the duplex template, introduces a complication situation (Figs. 2 and 5). The 3′ ^ 5′ strand is copied like RNA, while the 5′ ^ 3′ strand has to be copied in the opposite direction. It has been observed in all cases that simultaneous replication of both strands is accomplished by continuous copying of the 3′ ^ 5′ strand, also called the leading strand, while the 5′ ^ 3′ strand is copied after a brief delay when separation of the strands occur, so this nascent strand is called the lagging strand (Fig. 2). The leading strand can be synthesized continuously without interruption, while the lagging strand is synthesized dis-continuously after the leading strand is synthesized. The discontinuous fragments are also called Okazaki fragments, named after its discoverer.

C. Multiplicity of DNA and RNA Polymerases

Multiple DNA and RNA polymerases are present in both eukaryotes and prokaryotes, which evolved to fulfill distinct roles in the cell. In E. coli, DNA polymerases I (Pol I), II (Pol II), and III (Pol III) account for most DNA poly-merase activity. Pol I has the highest enzymatic activity and was the first DNA polymerase to be discovered by A Kornberg. However, Pol III is responsible for cellular DNA replication, while Pol I is involved in gap filling necessary during normal DNA replication (to fill in the space of degraded RNA primers) and also during repair of DNA damage. Pol II and two other DNA polymerases, Din B and UmuD/C, are responsible for replication of damaged DNA when it remains unrepaired.

Eukaryotic cells express five different DNA poly-merases, a, p, y, 5, and e, for normal DNA replication and repair. Pol a is involved in synthesis of primers for DNA replication; Pol p and possibly Pol e are involved in repair replication of damaged DNA. Pol 5 (and possibly Pol e) are responsible for replication of the nuclear genome. Pol y found in the mitochondria is responsible for replication of the mitochondrial genome. Several additional DNA polymerases recently identified and characterized are involved in replication of unrepaired damaged bases, like the E. coli DinB and UmuD/C (Table II).

E. coli has only one RNA polymerase, while eukary-otes have three distinct RNA polymerases, Pol I, Pol II, and Pol III, which transcribe different types of genes. RNA Pol I makes only ribosomal RNAs, which constitute the largest fraction of total RNA and, in fact, a significant fraction of the cellular mass. Pol III transcribes small RNAs, including transfer RNAs, which function as carriers of cognate amino acids and are required for protein synthesis. RNA Pol II transcribes all genes to generate mRNA, which encodes all proteins. Thus, this enzyme recognizes the most diverse group of genes. All of these RNA classes are initially synthesized as longer precursors that require extensive, often regulated, processing to yield the mature RNA product.

RNA and DNA polymerases encoded by virus and other episomal genomes are, in general, smaller and have fewer subunits than the cellular polymerases. Cellular poly-merase holoenzymes are rather complex with multiple

TABLE II Cellular DNA Polymerases

Prokaryote (E. coli)

In vivo function

Pol I

Nonreplicative removal of 5′ primer of Okazaki

fragments

Pol II

Nonreplicative, damage responsive polymerase

Pol III

Replicative synthesis

Din B

Lesion bypass DNA synthesis

UmuC

Lesion bypass DNA synthesis

Eukaryote

Pol a

RNA primer synthesis

Pol p

Repair synthesis

Pol S

Replicative (repair) synthesis

Pol e

Replicative (repair) synthesis

Pol Z

Damage bypass synthesis

Pol n

Damage bypass synthesis

Pol e

Damage bypass synthesis

Pol l

Damage bypass synthesis

Pol Y

Mitochondrial DNA synthesis

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