Essential Cell Biology 5th edition
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Essential Cell Biology 5th edition

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210 CHAPTER 6 DNA Replication and Repair5′incoming ribonucleosidetriphosphates3′Figure 6–17 RNA primers are synthesized by an RNA polymerasecalled primase, which uses a DNA strand as a template. Like DNApolymerase, primase synthesizes in the 5ʹ-to-3ʹ direction. Unlike DNApolymerase, however, primase can start a new polynucleotide chain byjoining together two nucleoside triphosphates without the need fora base-paired 3ʹ end as a starting point. Primase uses ribonucleosidetriphosphate rather than deoxyribonucleoside triphosphate.3′ HO5′ 3′5′ 3′3′5′3′5′templateDNA strandRNA primer3′ HOpreviousOkazakifragmentDNAlaggingstrandtemplate5′ECB5 e6.16-6.17previousRNAprimer5′PRIMASE JOINS TOGETHERTWO RIBONUCLEOTIDESPRIMASE SYNTHESIZESIN 5′-to-3′ DIRECTIONprimasenew RNA primersynthesized byprimase3′ 5′3′DNA POLYMERASE ADDSNUCLEOTIDES TO 3′ END OF NEWRNA PRIMER TO SYNTHESIZEOKAZAKI FRAGMENT5′3′ 5′3′stretch. DNA polymerase then adds a deoxyribonucleotide to the 3ʹ endof each new primer to produce another Okazaki fragment, and it willcontinue to elongate this fragment until it runs into the previously synthesizedRNA primer (Figure 6–18).To produce a continuous new DNA strand from the many separate piecesof nucleic acid made on the lagging strand, three additional enzymes areneeded. These act quickly to remove the RNA primer, replace it with DNA,and join the remaining DNA fragments together. A nuclease degradesthe RNA primer, a DNA polymerase called a repair polymerase replacesthe RNA primers with DNA (using the end of the adjacent Okazaki fragmentas its primer), and the enzyme DNA ligase joins the 5ʹ-phosphateend of one DNA fragment to the adjacent 3ʹ-hydroxyl end of the next(Figure 6–19). Because it was discovered first, the repair polymeraseinvolved in this process is often called DNA polymerase I; the polymerasethat carries out the bulk of DNA replication at the forks is known as DNApolymerase III.Unlike DNA polymerases I and III, primase does not proofread its work.As a result, primers frequently contain mistakes. But because primersare made of RNA instead of DNA, they stand out as “suspect copy” to beautomatically removed and replaced by DNA. The repair polymerase thatmakes this DNA, like the replicative polymerase, proofreads as it synthesizes.In this way, the cell’s replication machinery is able to begin newDNA strands and, at the same time, ensure that all of the DNA is copiedfaithfully.Proteins at a Replication Fork Cooperate to Forma Replication MachineDNA replication requires the cooperation of a large number of proteinsthat act in concert to synthesize new DNA. These proteins form part ofa remarkably complex replication machine. The first problem faced bythe replication machine is accessing the nucleotides that lie ahead ofthe replication fork and are thus buried within the double helix. For DNAreplication to occur, the double helix must be continuously pried apartso that the incoming nucleoside triphosphates can form base pairs with3′5′3′5′3′5′DNA POLYMERASE FINISHESOKAZAKI FRAGMENTPREVIOUS RNA PRIMER REMOVEDBY NUCLEASES AND REPLACED WITHDNA BY REPAIR POLYMERASE5′5′NICK SEALED BY DNA LIGASE5′3′3′3′Figure 6–18 Multiple enzymes are required to synthesize thelagging DNA strand. In eukaryotes, RNA primers are made at intervalsof about 200 nucleotides on the lagging strand, and each RNA primeris approximately 10 nucleotides long. These primers are extendedby a replicative DNA polymerase to produce Okazaki fragments. Theprimers are subsequently removed by nucleases that recognize theRNA strand in an RNA–DNA hybrid helix and degrade it; this leavesgaps that are filled in by a repair DNA polymerase that can proofreadas it fills in the gaps. The completed DNA fragments are finallyjoined together by an enzyme called DNA ligase, which catalyzes theformation of a phosphodiester bond between the 3ʹ-hydroxyl end ofone fragment and the 5ʹ-phosphate end of the next, thus linking upthe sugar–phosphate backbones. This nick-sealing reaction requires aninput of energy in the form of ATP (see Figure 6–19).

DNA Replication2115′ phosphateAATPhydrolyzedAAMP releasedAcontinuous DNA strand3′ 5′5′ 3′STEP 1STEP 2nicked DNA double helixFigure 6–19 DNA ligase joins together Okazaki fragments on the lagging strand during DNA synthesis. Theligase enzyme uses a molecule of ATP to activate the 5ʹ phosphate of one fragment (step 1) before forming a newbond with the 3ʹ hydroxyl of the other fragment (step 2).each template strand. Two types of replication proteins—DNA helicasesand single-strand DNA-binding proteins—cooperate to carry out this task.A helicase sits at the very front of the replication machine, where it usesthe energy of ATP hydrolysis to propel itself forward, prying apart thedouble helix as it speeds along the DNA (Figure 6–20 and Movie 6.2).Single-strand DNA-binding proteins then latch onto the single-strandedDNA exposed by the helicase, preventing the strands from re-formingbase pairs and keeping them in an elongated form so that they can serveas efficient templates.new Okazaki fragmentpreviousOkazakifragment(A)(B)leadingstrandtemplateleadingstrandtemplatenewly synthesizedDNA strandRNA primerlagging-strandtemplateDNA polymerase on lagging strand(just finishing an Okazaki fragment)start of nextOkazaki fragmentRNAprimernewlysynthesizedDNA strandnew OkazakifragmentECB5 e6.18-6.19sliding clampDNA polymerase onleading strandprimasesingle-strand DNAbindingproteinDNA polymeraseon lagging strand(just finishing anOkazaki fragment)DNA helicasenext Okazaki fragmentwill start hereparentalDNA helixlagging-strandtemplatepreviousOkazakifragmentparentalDNA helixFigure 6–20 DNA synthesis iscarried out by a group of proteinsthat act together as a replicationmachine. (A) DNA polymerases areheld on the leading- and laggingstrandtemplates by circular proteinclamps that allow the polymerases toslide. On the lagging-strand template,the clamp detaches each time thepolymerase completes an Okazakifragment. A clamp loader (not shown)is required to attach a sliding clampeach time a new Okazaki fragmentis synthesized. At the head of thefork, a DNA helicase unwinds thestrands of the parental DNA doublehelix. Single-strand DNA-bindingproteins keep the DNA strands apartto provide access for the primaseand polymerase. For simplicity, thisdiagram shows the proteins workingindependently; in the cell, they areheld together in a large replicationmachine, as shown in (B).(B) This diagram shows a currentview of how the replication proteinsare arranged when a replicationfork is moving. To generate thisstructure, the lagging strand shownin (A) has been folded to bring itsDNA polymerase in contact with theleading-strand DNA polymerase.This folding process also brings the3ʹ end of each completed Okazakifragment close to the start site forthe next Okazaki fragment. Becausethe lagging-strand DNA polymeraseis bound to the rest of the replicationproteins, the same polymerase canbe reused to synthesize successiveOkazaki fragments; in this diagram,the lagging-strand DNA polymeraseis about to let go of its completedOkazaki fragment and move to thenext RNA primer being synthesizedby the nearby primase. To watch thereplication complex in action, seeMovie 6.3 and Movie 6.4.

DNA Replication

211

5′ phosphate

A

ATP

hydrolyzed

A

AMP released

A

continuous DNA strand

3′ 5′

5′ 3′

STEP 1

STEP 2

nicked DNA double helix

Figure 6–19 DNA ligase joins together Okazaki fragments on the lagging strand during DNA synthesis. The

ligase enzyme uses a molecule of ATP to activate the 5ʹ phosphate of one fragment (step 1) before forming a new

bond with the 3ʹ hydroxyl of the other fragment (step 2).

each template strand. Two types of replication proteins—DNA helicases

and single-strand DNA-binding proteins—cooperate to carry out this task.

A helicase sits at the very front of the replication machine, where it uses

the energy of ATP hydrolysis to propel itself forward, prying apart the

double helix as it speeds along the DNA (Figure 6–20 and Movie 6.2).

Single-strand DNA-binding proteins then latch onto the single-stranded

DNA exposed by the helicase, preventing the strands from re-forming

base pairs and keeping them in an elongated form so that they can serve

as efficient templates.

new Okazaki fragment

previous

Okazaki

fragment

(A)

(B)

leadingstrand

template

leadingstrand

template

newly synthesized

DNA strand

RNA primer

lagging-strand

template

DNA polymerase on lagging strand

(just finishing an Okazaki fragment)

start of next

Okazaki fragment

RNA

primer

newly

synthesized

DNA strand

new Okazaki

fragment

ECB5 e6.18-6.19

sliding clamp

DNA polymerase on

leading strand

primase

single-strand DNAbinding

protein

DNA polymerase

on lagging strand

(just finishing an

Okazaki fragment)

DNA helicase

next Okazaki fragment

will start here

parental

DNA helix

lagging-strand

template

previous

Okazaki

fragment

parental

DNA helix

Figure 6–20 DNA synthesis is

carried out by a group of proteins

that act together as a replication

machine. (A) DNA polymerases are

held on the leading- and laggingstrand

templates by circular protein

clamps that allow the polymerases to

slide. On the lagging-strand template,

the clamp detaches each time the

polymerase completes an Okazaki

fragment. A clamp loader (not shown)

is required to attach a sliding clamp

each time a new Okazaki fragment

is synthesized. At the head of the

fork, a DNA helicase unwinds the

strands of the parental DNA double

helix. Single-strand DNA-binding

proteins keep the DNA strands apart

to provide access for the primase

and polymerase. For simplicity, this

diagram shows the proteins working

independently; in the cell, they are

held together in a large replication

machine, as shown in (B).

(B) This diagram shows a current

view of how the replication proteins

are arranged when a replication

fork is moving. To generate this

structure, the lagging strand shown

in (A) has been folded to bring its

DNA polymerase in contact with the

leading-strand DNA polymerase.

This folding process also brings the

3ʹ end of each completed Okazaki

fragment close to the start site for

the next Okazaki fragment. Because

the lagging-strand DNA polymerase

is bound to the rest of the replication

proteins, the same polymerase can

be reused to synthesize successive

Okazaki fragments; in this diagram,

the lagging-strand DNA polymerase

is about to let go of its completed

Okazaki fragment and move to the

next RNA primer being synthesized

by the nearby primase. To watch the

replication complex in action, see

Movie 6.3 and Movie 6.4.

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