1546 P r i m er
Primer
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1970
A primer is a short nucleic acid sequence that hybrid-
izes to one strand of DNA and provides a free 30 -OH
end at which a DNA polymerase starts synthesis of a
DNA chain.
See also: DNA Polymerases; Replication
Primer RNA
M A Griep
Copyright ß 2001 Academic Press
doi: 10.1006/rwgn.2001.1025
Definition
Primer RNA is RNA that initiates DNA synthesis.
Primers are required for DNA synthesis because no
known DNA polymerase is able to initiate poly-
nucleotide synthesis. DNA polymerases are special-
ized for elongating polynucleotide chains from their
available 30 -hydroxyl termini. In contrast, RNA poly-
merases can elongate and initiate polynucleotides.
Primases are special RNA polymerases that synthe-
size 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 gen-
ome utilizes RNA polymerase instead of primase to
initiate its DNA synthesis. In addition, the dominant
hypothesis concerning mitochondrial DNA replica-
tion initiation is that the mitochondrial RNA poly-
merase synthesizes a polymer that is not displaced
from the template. Then, the special RNase MRP
cleaves the ribopolymer at specific sites enabling the
exposed 30 -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 f29
covalently attach to both of the 50 -ends of linear duplex
DNA and provide a serine b-hydroxy group from
which a DNA polymerase elongates. Another ex-
ample is that many plasmids encode sequence-specific
nucleases which cleave one strand of the duplex to
create a 30 -hydroxyl for the host DNA polymerase.
An example of an animal virus is parvovirus, where the
30 -end of the parental strand forms a DNA hairpin and
becomes the primer of its complement.
Discontinuous DNA Synthesis and the
Primer RNA Hypothesis
After it was established in the mid-1960s that all
DNA and RNA polymerases catalyze polynucleotide
synthesis with 50 to 30 polarity, the Okazaki laboratory
performed pulse-chase experiments that helped to
resolve the paradox of the antiparallel nature of duplex
DNA. They discovered that two types of new DNA
were being synthesized. One was short (from 500 to
2000 nucleotides) while the other was much longer.
The short replicative intermediates, now referred to
as `Okazaki fragments,' represent discontinuously
synthesized DNA.
In bacteria and eukaryotes, discontinuous replica-
tion involves the following steps: (1) specific proteins
bind to the replicative origin; (2) a replicative helicase
is recruited to that complex; (3) the parental strands
are unwound; (4) primase is recruited and synthesizes
primer RNA on each of the two separated strands; (5)
a replicative DNA polymerase elongates from each of
these RNA primers to create two `leading' strands that
migrate away from each other (bidirectional replica-
tion) leaving the complementary single-stranded tem-
plate exposed; (6) helicase advances ahead of the
leading strand DNA polymerase to assist in duplex
unwinding; (7) single-stranded binding protein (SSB)
binds to the exposed single-strand template; (8) new
primer RNA molecules are synthesized complemen-
tary to the lagging strand template once every 500±
2000 nucleotides; and (9) another DNA polymerase
molecule elongates from those primer RNAs. Primer
RNA is then removed from the new strand to prevent
it from being incorporated into the chromosome.
The postreplicative excision processes are not direc-
tly coupled to discontinuous synthesis (Figure 1).
There are two enzymes that are able to remove the
majority, if not all, of the primer RNA. The first is a
50 -exonuclease and the second is RNase H (H stands
for RNA/DNA hybrid duplex). This ribonuclease has
the ability to hydrolyze ribopolymers that are co-
valently attached to deoxyribopolymers when the
latter is base-paired with another deoxyribonucleotide
polymer. After primer RNA has been fully removed
from the new strand, the resulting gap in the duplex is
filled in by a repair DNA polymerase. The last `nick'
in the backbone is closed by a DNA ligase. In bac-
teria, the primer-removing 50 -exonuclease is one of the
three domains of DNA polymerase I. In eukarya, it is
a free enzyme called Five0 EndoNuclease or Flap