Mary K. Campbell

Shawn O. Farrellinternational.cengage.com/

Chapter TenBiosynthesis of Nucleic Acids: Replication

Paul D. Adams • University of Arkansas

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Replication of DNA

• Naturally occurring DNA exists in single-stranded and double-stranded forms, both of which can exist

in linear and circular formsin linear and circular forms

• Difficult to generalize about all cases of DNA

replication

• We will study the replication of circular double-stranded DNA and then of linear double-stranded

DNADNA

• most of the details we discuss were first investigated

in prokaryotes, particularly E. coli

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Flow of Genetic Information in the Cell

• Mechanisms by which information is transferred in the cell is based on “Central Dogma”

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Prokaryotic Replication

• Challenges in duplication of circular double-stranded DNA

• achievement of continuous unwinding and • achievement of continuous unwinding and separation of the two DNA strands

• arotection of unwound portions from attack by

nucleases that attack single-stranded DNA

• synthesis of the DNA template from one 5’ -> 3’ strand and one 3’ -> 5’ strandstrand and one 3’ -> 5’ strand

• efficient protection from errors in replication

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Prokaryotic Replication (Cont’d)

• Replication involves separation of the two original strands and synthesis of two new daughter strands using the original strands strands using the original strands as templates

•• Semiconservative replication:Semiconservative replication:each daughter strand contains one template strand and one newly synthesized strand

• Incorporation of isotopic label as sole nitrogen source (15NH Cl)sole nitrogen source (15NH4Cl)

• Observed that 15N-DNA has a higher density than 14N-DNA, and the two can be separated by density-gradient ultracentrifugation

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Evidence for Semiconservative Replication

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Which Direction does Replication go?

• DNA double helix unwinds at a specific point called an

origin of replicationorigin of replication

• Polynucleotide chains are synthesized in both • Polynucleotide chains are synthesized in both

directions from the origin of replication; DNA replication is bidirectionalbidirectional in most organisms

• At each origin of replication, there are two replication replication forksforks, points at which new polynucleotide chains are

formed

• There is one origin of replication and two replication • There is one origin of replication and two replication

forks in the circular DNA of prokaryotes

• In replication of a eukaryotic chromosome, there are several origins of replication and two replication forks at each origin

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Bidirectional Replication

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DNA Polymerase Reaction

• The 3’-OH group at the end of the growing DNA chain acts as a nucleophile.

• The phosphorus adjacent to the sugar is attacked, • The phosphorus adjacent to the sugar is attacked, and then added to the growing chain.

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DNA Polymerase

• DNA is synthesized from its 5’ -> 3’ end (from the 3’ -> 5’ direction of the template)

• the leading strandleading strand is synthesized continuously in the 5’ -> 3’ direction toward the replication fork

• the lagging strandlagging strand is synthesized semidiscontinuously (Okazaki Okazaki • the lagging strandlagging strand is synthesized semidiscontinuously (Okazaki Okazaki fragments)fragments) also in the 5’ -> 3’ direction, but away from the replication fork

• lagging strand fragments are joined by the enzyme DNA ligaseDNA ligase

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Properties of DNA Polymerases

• There are at least five types of DNA polymeraseDNA polymerase (Pol) in E

coli, three of which have been studied extensively

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Function of DNA Polymerase

• DNA polymerase function has the following requirements:• all four deoxyribonucleoside triphosphates: dTTP,

dATP, dGTP, and dCTPdATP, dGTP, and dCTP• Mg2+

• an RNA primer - a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication

• DNA-Pol I: repair and patching of DNA (remove and • DNA-Pol I: repair and patching of DNA (remove and fill up primers in lagging strand)

• DNA-Pol III: responsible for the polymerization of the newly formed DNA strand

• DNA-Pol II, IV, and V: proofreading and repair enzymes

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The "real" The "real" replicativereplicative polymerase in E. colipolymerase in E. coli

�� It’s fast: up to 1,000 It’s fast: up to 1,000 dNTPsdNTPs added/sec/enzyme added/sec/enzyme

DNA Polymerase III DNA Polymerase III

�� It’s fast: up to 1,000 It’s fast: up to 1,000 dNTPsdNTPs added/sec/enzyme added/sec/enzyme

�� It’s highly It’s highly processiveprocessive: >500,000 : >500,000 dNTPsdNTPs added added before dissociatingbefore dissociating

�� It’s accurate: makes 1 error in 10It’s accurate: makes 1 error in 1077 dNTPsdNTPsadded, with proofreading, this gives a final added, with proofreading, this gives a final error rate of 1 in 10error rate of 1 in 1010 10 overall. overall. error rate of 1 in 10error rate of 1 in 1010 10 overall. overall.

IT’S COMPLICATED!!!

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Supercoiling and Replication

•• DNA gyraseDNA gyrase (class II topoisomerase) catalyzes reaction involving relaxed circular DNA:circular DNA:

• creates a nick in relaxed circular DNA

• a slight unwinding at the point of the nick introduces supercoiling

• the nick is resealed

• The energy required for this process is supplied by the hydrolysis of ATP to ADP and Pi

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Replication with Supercoiled DNA

• Replication of supercoiled circular DNA

•• DNA DNA gyrasegyrase has different role here. Ithas different role here. It introduces a nick in supercoiled DNA

• a swivel point is created at the site of the nick• a swivel point is created at the site of the nick

• the gyrase opens and reseals the swivel point in advance of the replication fork

• the newly synthesized DNA automatically assumes the supercoiled form because it does not have the nick at the swivel point

•• helicasehelicase, a helix-destabilizing protein, promotes unwinding by binding at the replication fork

• single-stranded binding (SSB) protein stabilizes single-stranded regions by binding tightly to them

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Primase Reaction

• The primase reaction

• RNA serves as a primer in DNA replication

• primer activity first observed in-vivo.

•• PrimasePrimase -- catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence

• Synthesis and linking of new DNA strands

• begun by DNA polymerase III

• the newly formed DNA is linked to the 3’-OH of the • the newly formed DNA is linked to the 3’-OH of the RNA primer

• as the replication fork moves away, the RNA primer is removed by DNA polymerase I

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Replication Fork General Features

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1. Unwinding of parental duplex by helicase with help of gyrase and elongation of leading

strand by DNA polymerase III

Overview of DNA Replication Overview of DNA Replication Overview of DNA Replication Overview of DNA Replication Synthesis of the lagging strand

strand by DNA polymerase III expose single-strand region in front of lagging strand

2. Primase synthesizes RNA primer

3. Polymerase III extends DNA Okazaki fragmentfrom primer

4. Polymerase I eliminates downstream RNA primer by nick translation

5. DNA ligase ligates Okazaki fragment to rest of lagging strand

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Proofreading and Repair

• DNA replication takes place only once each generation in each cell

• Errors in replication (mutations) occur spontaneously only • Errors in replication (mutations) occur spontaneously only once in every 109 to 1010 base pairs

• Can be lethal to organisms

• Proofreading - the removal of incorrect nucleotides immediately after they are added to the growing DNA immediately after they are added to the growing DNA during replication (Figure 10.10)

• Errors in hydrogen bonding lead to errors in a growing DNA chain once in every 104 to 105 base pairs

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Proofreading Improves Replication Fidelity

•• CutCut--andand--patchpatch catalyzed by Pol I: cutting is removal of the RNA primer and patching is incorporation of the required deoxynucleotides

•• Nick translationNick translation: Pol I removes RNA primer or DNA •• Nick translationNick translation: Pol I removes RNA primer or DNA mistakes as it moves along the DNA and then fills in behind it with its polymerase activity

•• Mismatch repair:Mismatch repair: enzymes recognize that two bases are incorrectly paired, the area of mismatch is removed, and the area replicated again

•• Base excision repair:Base excision repair: a damaged base is removed by DNA glycosylase leaving an AP site; the sugar and DNA glycosylase leaving an AP site; the sugar and phosphate are removed along with several more bases, and then Pol I fills the gap

• Nucleotide excition repair: damaged nucleotides that lead to deformed DNA structures are removed as part of a large section that contain the deformed structure

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DNA Polymerase Repair

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Mismatch Repair in Prokaryotes

• Mechanisms of mismatch repair encompass:

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Base excision repair

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Nucleotide excision repair

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DNA Recombination

• Genetic Recombination- When genetic information is rearranged to form new associations

• Homologous - Reactions between homologous sequences

• Nonhomologous- Different nucleotide sequences recombinerecombine

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Recombination

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Eukaryotic Replication

• Not as understood as prokaryotic. Due in no

small part to higher level small part to higher level of complexity.

• Cell growth and division divided into phases: M,

G1, S, and G2G1, S, and G2

• DNA replication takes place during S phase

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Eukaryotic Replication

• Best understood model for control of

eukaryotic replication eukaryotic replication is from yeast.

• DNA replication initiated by

chromosomes that chromosomes that have reached the G1

phase

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Eukaryotic Replication

• ORC – origin recognition

complexcomplex

• RAP – replication activator

protein

• RLF – replication licensing

factors

• CDK – cyclin dependent • CDK – cyclin dependent

protein kinases

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Eukaryotic DNA Polymerase

• At least 15 different polymerases are present in eukaryotes (5 have been studied more extensively)

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Structure of the PCNA Homotrimer

• PCNA (proliferating cell nuclear antigen) is the eukaryotic equivalent of the part of Pol III that functions as a sliding clamp (β).

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The Eukaryotic Replication Fork

(single stranded binding protein)

(replication factor C)

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The Eukaryotic Replication Fork – initiation of replication (in yeast)

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The Eukaryotic Replication Fork

• The general features of DNA replication in eukaryotes are

similar to those in prokaryotes. Differences summarized in

Table 10.5.

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Telomerase and Cancer (Biochemical Connections)

• Replication of linear DNA molecules poses particular problems at the ends of the molecules

• Ends of eukaryotic chromosomes called telomeres

• Telomere- series of repeated DNA sequences

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