The Growing Point Paradox and Discontinuous DNA Synthesis

Studies of replicating DNA molecules by autoradiography and electron

microscopy indicate that the two progeny strands being synthesized at each
replicating fork are being extended in the same overall direction, at least on the
macromolecular level. Since the complementary strands of a double helix have
opposite polarity, this mean that synthesis is occurring at the 5’ end of one strand
(or 3’-5’) and the 3’ end of the other strand (5’-3’). As discussed in the preceding
sections, however, all known polymerases have an absolute requirement for a free
3’ hydroxyl, they only carry out 5’-3’ synthesis. This paradox exsted for many
years during which biochemists searched in vain for new polymerases that could
carry out 3’-5’ synthesis. No such polymerase has yet been found. Instead, storng
evidence has accumulated indicating that all synthesis occurs in the 5’-3’
direction. The resolution of the paradox resulted from the demonstration that the
synthesis of one DNA strand is discontinuous.

Autoradiography and electron microscopy show that the two nascent

DNA strands being synthezised at each replicating fork are being extended in the
same direction at the macromolecular level. Since the complementary strands of a
DNA double helix have opposite chemical polarity, one strand is being extended
in an overall 3’-5’ direction. At the molecular level, however, synthesis is actually
occuring in opposite directions strand are being synthesized in the 5’-3’ direction.
The strands being extended in the overal 3’-5’ direction grow by the synthesis of
short segments (synthesized 5’-3’) and the subsequent joining of these short
segments by polynucleotide ligase. The evidence for this discontinuous mode of
DNA replication has come from studies in which intermediates in DNA synthesis
were radioactively labeled by growth of cells for very short periods of time in
medium containing (H3) thymidine (pulse labeling). When e coli cells were pulse
labeled for 15 seconds for example, all the label was found in a small pieces,
1000-2000 nucleotides long. These small pieces or segments of DNA, often called
“Okazaki fragments” after R. Okazaki, who first identified them, are smaller,
about 100-200 nucleotides long, in eukaryotes. When longer pulse labeling
periods are used, more of the label is recovered in large DNA molecules probably
the size of molecules containing all the DNA present in intact chromosomes. In
short pulse labeling periods, the radioactivity present in short DNA “fragments”
becomes incorporated in chromosome sized DNA molecules during subsequent
growth of the cells on medium containing nonradioactive thymidine. This is
important because it indicates that the “Okizaki fragments” are true intermediates
in DNA synthesis rather than some kind of metabolic by product. Extensive
evidence has shown that DNA synthesis is continuous for the strand growing in
the overall 5’-3’ direction (sometimes called the “labeling” strand) and is
discontinuous for the strand growing in the overall 3’-5’ direction (sometimes
called “lagging” strand).
 Initiation and the “Primer Problem”

As has been emphasized earlier, all known DNA polymerases have an

absolute requirement for a free 3’ OH on a DNA primer plus an appropriate DNA
template strand for activity. Thus, no known DNA polymerase can initiate the
synthesis of a new strand of DNA. Since the synthesis of each “Okazaki
fragment” requires an initiation event, an efficient mechanism of chain initiation
is essential for ongoing DNA replication. RNA polymerase a complex enzime that
catalyzes the synthesis of RNA molecules from DNA template, has long been
known to be capable an initiating the synthesis of new RNA chains at specifcsites
on the DNA. When this occurs, an RNA-DNA hybrid is formed in which the
nascent RNA is hydrogen bonded to the DNA template. Since DNA polymerases
are capable of extending polynucleotide chains containing an RNA primer with a
free 3’OH, scientists in several laboratories began testing the idea thet DNA
synthesis is initiated by RNA primers. There is now definitive evidence
supporting the proposal that DNA synthesis is “primed” by short segments of
RNA, which are later removed by a 5’-3’ exonuclease and replaced by DNA prior
to covalent sealing by and replaced by DNA prior to covalent sealing by
polinucleotide ligase. In e coli, the RNA primers are excised by the 5’-3’
exonuclease activity of DNA polymerase 1. This occurs simultaneously with the
synthesis of new DNA strands (replacing the excised RNA primer strands) by the
5’-3’ polymerase activity of this enzyme.

The synthesis of the RNA primers is catalyzed by enzimes called

primases, which have properties quite distintc from those of the RNA
polymerases. The e coli primase is the product of the dnaG gene. In prokaryotes,
the RNA primers are 10-60 nucleotides in length. In eukaryotes they are quite
short, about 10 nucleotides long. The use of RNA primers is almost certainly the
most common mechanism used to initiate DNA synthesis. Nevertheless, certain
viruses appear to have evolved quite different mechanism for the innitiation of
DNA synthesis.

The complete “replication apparatus” is complex

When watson and crick worked out the double helix structure of DNA,
they immediately recognized that the complementary nature of the two strands
provided a simple basis for the faithful duplication of genetic material. Meselson
and stahl’s demonstration of the semiconservative replication of the e coli
chromosome solidified the concept that the two strands of the double helix
unwind and serve as templates for the synthesis of complemetary strands. Thus, a
parental double helix direct the synthesis of two identical progeny double helices.
Komberg’s isolation of an enzyme, DNA polymerase I, capable of synthesizing
DNA in vitro appeared to provide the final link in what was thought to be an
elegantly simple mechanism for the replication of the genetic material but such
was not case. Twenty years later, scientists are still trying to work out the details
of the mechanism of DNA replication.

DNA replication is complex. It is carried out by a multienzyme complex,

often called the replication apparatus or the repliosome. In eukaryotes, the
components of the replication machinery are just begining to be identified. Even
in prokaryotes, DNA replication requires many different proteins, and the details
of how some of these proteins function in DNA replication are still being
invetigated today. For example, DNA replication in e coli requires at least two
dozen different gene products. Many of these gene product have been purified an
their roles in DNA replication studied in vitro. The involvement of some of these
e coli proteins in DNA replication, it is intended to illustrate the complexity of the
replication process rather than to illustrate the specific roles of the individual gene
products.

First, the two complementary strands of the parental double helix have to
be unwound and separated so that each can serve as a template for the synthesis of
a new daughter strand. Unwinding and movement of the replication fork occur
processively with the strands being transiently unwound ahead of the fork as it
moves along the chromosome. Three different types of proteins appear to
contribute to unwinding the strands of double helices. (1) DNA unwinding
proteins or DNA belicases are dirctly involved in catalyzing the unwinding of the
double helices. In e coli, two different helicases are involved. One helicase, the
product of the rep gene, binds to and stimulates separationss of the strand that has
3’ to 5’ polarity in the direction of replication fork movement. The other helicase
(exact identity still uncertain) binds to and assists unwinding of the strand that has
5’ to 3’ polarity in the direction that the fork is moving. (2) DNA single strand
binding proteins (SSBPs) bind tightly to single stranded regions of DNA produced
by the action of the helicases and help stabilize the extended single stranded
templates needed fot polymerization. The SSBPs bind to DNA as tretamers, and
their binding exhibits cooperativity (the binding of one tetramer stimulates the
binding of additional tetramers to adjacent segments of single stranded DNA).
Single stranded DNA that is saturated with bound SSBPs replicates over 100
times faster than uncomplexed single strands of DNA secondary structures that
interfere with the movement of DNA polymerases or other components of the
replications complex along the molecule in the normal processive manner. (3)
finally, DNA gyrases, which catalyze the formation of negative supercoils in
DNA, are essential for replication and are believed to play a key role in the
unwinding process. Supercoiling has been proposed to hel “drive” the unwinding
process, however, we still do not know how this works. Very recently, it has been
suggested that DNA gyrase may function by removing positive supercoils that
accumulate in front of the replication forks as the helicases unwind the double
helicases. In any case, DNA gyrases are essential for DNA replication and
somehow maintain pre and post repliacative DNAs in the propertopological
structures.

Nascent DNA strands are then initiated by the use of RNA primers by the
mechanism discussed earlier. Synthesis of the RNA primers is catalyzed by a
special class of enzymes called primases. Primase activity requires the formation
of a complex of primase and at least six other proteins, this complex is called
primosome. In addition to primase, the primosome contains prepriming proteins
tentatively designated proteins i, n, n+ and n plus the products of genes dnaB and
dnaC. The primosome carries out the initial priming reaction for the loading
strand (the strand extended continuously in the overall 3’ to 5’ direction but 5’ to
3’ at the molecular level). The functions of the individual proteins in the
primosome are still uncertain.

The covalent extension of the primed DNA chains during chromosome

replication in e coli is carried out by DNA polymerase III. Unlike DNA
polymerase I of e coli (which is a single polypeptides). DNA polymerase III is a
complex enzyme centaining seven different polypeptides an all of these
polypeptides must be present for proper replicative function. The 5’ to 3’
polymerase activity and the 5’ to 3’ exonuclease activity are both present on the α
polypeptide of DNA polymerase III. The 3’ to 5’ proofreading activity of
polymerase III is present on the ϵ polypeptide. The functions of the other subunits
are still uncertian. Subsequent to DNA polymerase III activity at the replication
fork, DNA polymerase I catalyzes the removal of the RNA primers by the
concerted action of its 5’ to 3’ exonuclease activity and its 5’ to 3’ polymerase
activity, and DNA ligase catalyze covalent closure of the resulting single stranded
“nick”.

Several components essential for DNA replication have been identified

genetically, taht is, e coli strains carrying mutations (heritable changes in the
genetic material) the result in the inability to replicate DNA under certain
conditions (usually high temperature) have been identified. When these mutations
were characterized genetically, they were found to idntify a set of genes
(designated dnaA, dnaB, dnaC, etc) whose products of the some of these genes are
known. For example, dnaF, dnaN, dnaX and dnaZ code for four of the seven
subunits (polypeptides) of the complete DNA polymerase III enzyme, and dnaG
specifies the primase. The product and functions of the others are still unknown.
Other components of the replication enzymes ( some of the subunits of DNA
polymerase III) were discovered by biochemical analyses, and genes that encode
these proteins have still not been identified.
 It is hoped that exact functions of the many gene products involved in
replication will be worked out during the next few years. Attempts to isolate intact
functional replisomes, however, heve been ;lagerly unsuccesfull. Reconstitution
of subcomplexes of replication aparatuses from purified proteins has been more
successful. This is undoubtedly a result of the fact that the complexes are held
together by relatively weak protein-protein interactions, which are disruped during
the isolation procedured. In addition, replication cmoplexes may be membrane
bound and require membrane structures for their assembly. There is considerable
evidence that replication forks are assosiated with the cell membrane in
prokaryotes and with the nuclear envelope in eukaryotes.

For excellent, more detailed accounts of replication an the components of

the replication apparatus, the reader is referred to Komberg’s DNA replication and
1982 supplement to DNA replication.