Primer

Primer LJ Reha-Krantz and L Zhang, University of Alberta, Edmonton, AB, Canada

© 2013 Elsevier Inc. All rights reserved.

This article is a revision of the previous edition article, volume 3, p 1546, © 2001, Elsevier Inc.

Glossary DNA polymerase An enzyme that copies or replicates the DNA genome of an organism. DNA polymerases synthesize a complementary DNA copy of a DNA strand using the four building blocks of DNA – deoxyribonucleoside triphosphates. Primase An enzyme that synthesizes RNA primers for the initiation of DNA replication.

The Initiation of DNA Replication: In Vitro Reactions An important difference between DNA polymerases that repli­ cate DNA and RNA polymerases that transcribe DNA is that RNA polymerases initiate RNA synthesis de novo, which means that the first and all subsequent ribonucleotides incorporated into the RNA transcript are added by the RNA polymerase (Figure 1(a)). By contrast, DNA polymerases require help to start or prime the initiation reaction. In many cases, the primer is a complimentary RNA or DNA that is annealed to the tem­ plate strand, which can then be extended by a DNA polymerase (Figure 1(b)). Although the first DNA polymerase, DNA pol I, was discovered in Escherichia coli in 1959 by A. Kornberg, the mechanism of initiation of DNA replication was not known for several years. Kornberg and colleagues demonstrated in 1967 that DNA pol I could replicate a complimentary copy of the single-stranded, circular φX174 viral genome in reactions that required the four building blocks of DNA – the deoxynu­ cleoside triphosphates (dATP, dCTP, dGTP, and dTTP) and magnesium, which is necessary for DNA polymerase activity. In addition, a small amount of boiled E. coli extract was required for the initiation of DNA synthesis. The initiation factor in the extract was determined in 1968 to be short, single-stranded DNAs because enzymes that degraded single-stranded DNA, but not enzymes that degraded proteins or RNA, destroyed the initiation activity. These studies demon­ strated that random, complimentary single-stranded DNAs were sufficient to prime DNA replication in test tube reactions (in vitro), but random priming is not observed in nature (in vivo); instead, DNA replication initiates at specific sites called origins of replication.

The Initiation of DNA Replication In Vivo Because RNA polymerases initiate the synthesis of RNA with­ out the assistance of a primer (Figure 1(a)), A. Kornberg proposed that RNA polymerases could synthesize RNAs in origin regions that were then extended by DNA polymerases (Figure 1(b)). RNA polymerase-dependent priming of DNA

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Primosome A complex of a primase and a helicase that synthesizes RNA primers for the initiation of DNA replication. RNA polymerase An enzyme that synthesizes a complementary RNA copy of a DNA strand using the four building blocks of RNA – ribonucleoside triphosphates.

replication is required for replication of the single-stranded, circular DNA genome of bacteriophage M13, a virus that infects E. coli (Figure 2(a)). Rifampicin, an inhibitor of bacterial RNA polymerase, prevents phage M13 DNA replication in vivo. RNA polymerase-dependent priming was also demonstrated in vitro. RNA polymerase is also required to synthesize specific RNA primers for the initiation of DNA replication for ColE1 (pBR322) plasmids, but RNA polymerase-dependent priming is not the norm. Instead, many organisms encode enzymes called ‘primases’ that synthesize short, complementary RNAs that are used for priming DNA replication (Figure 2(b)). Primases associate with additional proteins to form primo­ somes, which are mobile priming machines that synthesize RNA primers for leading- and lagging-strand DNA replication. In E. coli, DNA replication is initiated at a single origin of replication in the �4700-kb circular, double-stranded genome (Figure 2(b)). DNA initiation requires a specific initiation protein (DnaA) that binds to the origin. The DnaB helicase unzips the DNA strands and then associates with the primase (DnaG), which synthesizes short RNA primers that are extended by the DNA pol III holoenzyme. Leading-strand replication needs to be primed once, which takes place in the origin region, but lagging-strand replication needs repeated priming, which generates short DNA fragments called ‘Okazaki fragments’. Lagging-strand replication is discontinuous compared to leading-strand replication because the two strands of duplex DNA are of opposite polarities and DNA replication is only in the 5′–3′ direction as explained in detail elsewhere in this encyclopedia. The DnaB/DnaG primosome synthesizes RNA primers for both leading- and lagging-strand replication. Eukaryotes follow a similar mechanism for priming DNA replication as shown for yeast (Figure 2(b)). The mini chromo­ some maintenance helicase complex (MCM) helicase unzips the DNA strands. RNA primers are synthesized by a four-subunit complex that contains the primase and DNA polymerase α. Primase synthesizes a short complementary RNA that is extended by DNA polymerase α. The RNA–DNA primer is then extended by DNA polymerase ε on the leading strand and by DNA polymerase δ on the lagging strand. There are several additional DNA polymerase priming mechanisms that are used by viruses, bacteriophage, and for

Brenner’s Encyclopedia of Genetics, 2nd edition, Volume 5

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(a) RNA polymerase transcription initation

(a) RNA polymerase priming of phage M13 DNA replication E.coli DNA pol III

A T

5′

T G T

Pr

G

RNA pol

A ppp OH A T A U U G U C T A A C A

3′

holoenzyme

im

er

M13ssDNA Rifampicin

5′

E.coli pol I

RNA removal and gap repair

Direction of transcription (b) DNA polymerase primer extension primer

OH

DNA ligase

OH

3′

P

Seals nick

Template strand 5′

(b) Primase-dependent priming

OH

E.coli

3′

Origin

3′

5′

OH

primer

Lagging strand

3′ Okazaki fragment

Yeast 3′

Direction of replication Figure 1 Initiation of transcription (a) compared to the initiation of DNA replication (b).

Primer: pppAG(N) 8-10 Primosome: DnaG-DnaB

Pol ε

PCN

A

plasmid replication. Terminal proteins are used for initiation of replication of the linear genomes of adenovirus and the Bacillus subtilis phage φ29. The phage φ29 terminal protein associates with the φ29 DNA polymerase to form a priming complex that associates with the ends of the phage chromosome. The DNA polymerase catalyzes a covalent bond between dAMP and an essential serine residue in the terminal protein (Figure 2(c)). Once DNA replication is initiated, highly processive DNA synthesis continues until the template strand is fully replicated; the terminal protein remains attached to the 5′-end. The second strand is replicated in the same way; thus, two viral genomes are produced, each with one parental and one newly synthe­ sized strand. Two RNA genomes are packaged in retrovirus particles along with specific host transfer RNAs (tRNAs) that are used to prime DNA replication by the viral reverse transcriptase (Figure 2(d)). DNA polymerases that use RNA as a template are called ‘reverse transcriptases’. Once DNA replication extends past the 5′ R (repeat) region, removal of this region of the RNA template produces a single-stranded DNA that is used to prime synthesis from the 3′ R region of the second viral genome. This process generates a complete viral DNA strand. DNA primers can also be generated by nicking one strand of duplex DNA. Several plasmids and bacteriophage use site-specific endonucleases to nick one strand of DNA in the origin region. DNA polymerases use the 3′-end of the nick to prime DNA replication. A helicase displaces the complementary DNA strand ahead of the replicating DNA polymerase. Recombination can also provide a DNA primer for DNA replication. During bacteriophage T4 late DNA replication, recombination processes generate 3′-single-stranded DNAs that invade complimentary regions of duplex DNA in other genomes present in the infected

5′ 3′

Leading strand

5′

RPA δ Pol

pol α­ primase

Okazaki fragment

5’ 3’

MCM Primer: (N) ppp(A/G)(N) 7-9 6 RNA DNA

5′

(c) Terminal protein priming 3′ 3′ Terminal protein Initiation DNA pol 3′

Ser-A-A-OH 3′

HO-A-A-Ser

Elongation

Ser-A-ATT 3′

3′ TT A-A-Ser

(d) tRNA priming tRNA HO-3′ 5′

R

5′

PBS

Reverse transcriptase 5′

Viral RNA 3′

R DNA synthesis by Reverse transcriptase

HO-3′ 5′

3′ Partial RNA removal

5’

5’

Reposition of single-stranded DNA 5′ HO- 3’ 3′ Full length DNA synthesis by reverse transcriptase 5′ 3′

Figure 2 Primers used for the initiation of DNA replication.

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bacterial host. The recombination intermediates are used as primers for further DNA replication.

See also: Discontinuous Replication; Leading Strand; Okazaki Fragment; Replication Fork.

Summary DNA polymerases cannot initiate synthesis of DNA chains in the absence of a priming device. Although DNA polymerases can readily extend short DNA primers that are randomly annealed to the template strand in vitro, specific priming is observed in vivo. In vivo primers may be the following: RNA synthesized by RNA polymerases, by primases or the RNA is pre-formed (tRNA); DNA produced by combination of an initial RNA primer and then extension by a DNA polymerase as observed for the dual action of primase and DNA polymerase α in eukaryotes; DNA primers produced by specific nicking of one strand of DNA or by recombination; and terminal proteins.

Further Reading Brutlag D, Schekman R, and Kornberg A (1971) A possible role for RNA polymerase in the initiation of M13 DNA synthesis. Proceedings of the National Academy of Sciences of United States of America 68: 2826–2829. Goulian M (1968) Incorporation of oligodeoxynucleotides into DNA. Proceedings of the National Academy of Sciences USA 61: 284–291. Goulian M and Kornberg A (1967) Enzymatic synthesis of DNA, XXIII. Synthesis of circular replicative form of phage φX174 DNA. Proceedings of the National Academy of Sciences USA 58: 1723–1730. Méndez J, Blanco L, and Salas M (1997) Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase. EMBO Journal 16: 2519–2522.