Chapter Ten Biosynthesis of Nucleic Acids: Replication Paul D. - - PDF document

Mary K. Campbell Shawn O. Farrell

• Chapter Ten

Biosynthesis 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 forms in 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 DNA DNA

• 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’ strand strand 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
• rigin of replication
• rigin of replication
• Polynucleotide chains are synthesized in both
• Polynucleotide chains are synthesized in both

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

• At each origin of replication, there are two replication

replication forks forks, 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 strand

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

• the lagging strand

lagging strand is synthesized semidiscontinuously (Okazaki Okazaki

• the lagging strand

lagging 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 ligase

DNA ligase

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Properties of DNA Polymerases

• There are at least five types of DNA polymerase

DNA 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 dCTP dATP, 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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DNA Polymerase III DNA Polymerase III

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Supercoiling and Replication

• DNA gyrase

DNA 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 gyrase gyrase has different role here. It has 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

• helicase

helicase, 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.
• Primase

Primase - 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

• 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 fragment from 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
• nce 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

• Cut

Cut-and and-patch patch catalyzed by Pol I: cutting is removal of the RNA primer and patching is incorporation of the required deoxynucleotides

• Nick translation

Nick translation: Pol I removes RNA primer or DNA

• Nick translation

Nick 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 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

recombine recombine

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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

• f complexity.
• Cell growth and division

divided into phases: M, G1, S, and G2 G1, 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

complex complex

• 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
• f 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

• f 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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