Essential Cell Biology 5th edition

212 CHAPTER 6 DNA Replication and Repair
QUESTION 6–2
Discuss the following statement:
“Primase is a sloppy enzyme that
makes many mistakes. Eventually,
the RNA primers it makes are
removed and replaced with DNA
synthesized by a polymerase with
higher fidelity. This is wasteful. It
would be more energy-efficient
if a DNA polymerase were used
to make an accurate primer in
the first place.”
This localized unwinding of the DNA double helix itself presents a problem.
As the helicase moves forward, prying open the double helix, the
DNA ahead of the fork gets wound more tightly. This excess twisting in
front of the replication fork creates tension in the DNA that—if allowed
to build—makes unwinding the double helix increasingly difficult and
ultimately impedes the forward movement of the replication machinery
(Figure 6–21A). Enzymes called DNA topoisomerases relieve this tension.
A DNA topoisomerase produces a transient, single-strand nick in
the DNA backbone, which temporarily releases the built-up tension; the
enzyme then reseals the nick before falling off the DNA (Figure 6–21B).
Back at the replication fork, an additional protein, called a sliding clamp,
keeps DNA polymerase firmly attached to the template while it is synthesizing
new strands of DNA. Left on their own, most DNA polymerase
molecules will synthesize only a short string of nucleotides before falling
off the DNA template strand. The sliding clamp forms a ring around the
newly formed DNA double helix and, by tightly gripping the polymerase,
allows the enzyme to move along the template strand without falling off
as it synthesizes new DNA (see Figure 6–20A and Movie 6.5).
Assembly of the clamp around DNA requires the activity of another replication
protein, the clamp loader, which hydrolyzes ATP each time it locks
a sliding clamp around a newly formed DNA double helix. This loading
needs to occur only once per replication cycle on the leading strand; on
the lagging strand, however, the clamp is removed and then reattached
each time a new Okazaki fragment is made. In bacteria, this happens
approximately once per second.
Most of the proteins involved in DNA replication are held together in
a large multienzyme complex that moves as a unit along the parental
DNA double helix, enabling DNA to be synthesized on both strands in a
coordinated manner. This complex can be likened to a miniature sewing
machine composed of protein parts and powered by nucleoside triphosphate
hydrolysis (Figure 6–20B). The proteins involved in DNA replication
are listed in Table 6–1.
leading-strand
template
DNA supercoil
3′
3′
Figure 6–21 DNA topoisomerases
relieve the tension that builds up in
front of a replication fork. (A) As a
DNA helicase moves forward, unwinding
the DNA double helix, it generates a
section of overwound DNA ahead of it.
Tension builds up because the rest of
the chromosome (shown in brown) is too
large to rotate fast enough to relieve the
buildup of torsional stress. The broken
bars represent approximately 20 turns
of DNA. (B) Some of this torsional stress
is relieved by additional coiling of the
DNA double helix to form supercoils.
(C) DNA topoisomerases relieve this stress
by generating temporary nicks in the
DNA, which allow rapid rotation around
the single strands opposite the nicks.
5′
DNA helicase
(A)
3′
5′
(C)
in the absence of topoisomerase, the DNA cannot
rapidly rotate, and torsional stress builds up
(B)
5′
lagging-strand
template
DNA topoisomerase creates transient
single-strand break
some torsional stress is relieved by
DNA supercoiling
site of
free rotation
torsional stress ahead of the helicase relieved by free rotation of DNA around the
phosphodiester bond opposite the single-strand break; the same DNA topoisomerase
that produced the break reseals it