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DNA Is Reproduced by Semiconservative Replication CHAPTER 10: DNA Replication & Recombination
► The complementarity of DNA strands allows each strand to serve as a DNA Replication in Yeast
template for synthesis of the other. ► Yeast has between 250 and 400 replicons per genome.
► Three possible modes of DNA replication were postulated: ► Some mammalian cells have as many as 2500 replicons per genome!
• conservative ► Yeast autonomously replicating sequences (ARSs) contain an 11-bp
► The Meselson-Stahl experiment consensus sequence involved in efficient initiation.
• semiconservative demonstrated that:
• dispersive ► Not all origins of replication are activated all at once. Rather, clusters
• DNA replication is semiconservative in of about 20-80 replicons are activated sequentially throughout the S phase
► The Taylor- prokaryotes
Woods-Hughes until all DNA is replicated.
• each new DNA molecule consists of one old How does DNA polymerase finds such ARSs among so much DNA?

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experiment strand and one newly synthesized strand
demonstrated that ► In the G1 phase of cell cycle, the ARSs are initially bound by a group of proteins to
DNA replication is form the origin recognition complex (ORC).
DNA Replication Directionality
semiconservative in ► Among other kinases (initiation proteins), these proteins at the ORC form a pre-
eukaryotes. ► DNA replication begins at the origin of replication replication complex that becomes accessible to DNA polymerase once kinases are
(Ori) and is bidirectional rather than unidirectional. activated.
► A replicon is the length of DNA that is replicated ► Phosphorylation is also important in preventing the reformation of the pre-replication
following one initiation event at a single origin.

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complex once DNA replication is completed
DNA Synthesis in Bacteria Involves Five Polymerases, as well as Other Enzymes
► DNA polymerase I catalyzes DNA synthesis and requires a DNA template and all four dNTPs. DNA Replication in Eukaryotes
► However, the ability of E. coli cells with mutant DNA polymerase I to replicate normally ► Three DNA polymerases are involved in replicating
suggested the existence of other DNA polymerases that can catalyze DNA polymerization. nuclear DNA.
► Now, we know that DNA polymerases I, II, III, IV, and V exist in prokaryotes. ► One DNA polymerase is involved in replicating
► Only DNA polymerases I, IV, and V, however, are involved in various aspects of repair of DNA mitochondrial DNA.
that was damaged due to external forces (e.g. UV light). ► Others are involved in DNA repair processes.
► Chain elongation occurs in the 5' to 3' direction by ► Pol and are the major forms of the enzyme
addition of one nucleotide at a time to the 3' end. involved in initiation and elongation.
► As the nucleotide is added, the two terminal phosphates ► Pol possesses low processivity, a term that reflects the length
(pp) are cleaved off. of DNA that is synthesized by an enzyme before it dissociates
► DNA polymerases I, II, and III can elongate an existing from the template.
DNA strand (called a primer) but CANNOT initiate DNA ► Pol functions in synthesis of the RNA primers during
synthesis. initiation on the leading and lagging strands.
► All DNA polymerases I, II, and III possess 3' to 5' ► Polymerase switching occurs, and Pol replaced by Pol for
exonuclease activity. elongation.
► However, only DNA polymerase I demonstrates 5' to 3' ► RNA primer removal is also performed by Pol
exonuclease activity. ► Telomeres at the ends of linear chromosomes consist of long stretches of
► DNA polymerase III is the enzyme responsible for the 5' to 3' polymerization essential in vivo. short repeating sequences bound to specific telomere- associated proteins and
► Its 3' to 5' exonuclease activity allows proofreading. preserve the integrity and stability of chromosomes.
► DNA polymerase III is a holoenzyme that has 10 subunits. ► Lagging strand synthesis at the end of the chromosome is a problem because
► The holoenzyme and several other proteins at the replication fork together form a huge once the RNA primer is removed, there is no free 3'- hydroxyl group from which
complex known as the replisome. to elongate.
► Polymerase I is believed to be responsible for: removing the primer and the synthesis that ► Telomerase directs synthesis of the telomere repeat sequence to fill the gap.

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fills gaps produced during synthesis ► The enzyme telomerase directs synthesis of the 5’-TTGGGG-3’ sequences,
resulting in the formation of a hairpin structure. The gap can now be filled, and,

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following cleavage of the hairpin structure, the process prevents the creation of a
gap during replication of the ends of linear chromosomes.
Many Complex Tasks Must Be Performed during DNA Replication ► Genetic exchange at equivalent positions along two chromosomes with substantial
► There are seven key issues that must be resolved during DNA replication: DNA sequence homology is referred to as general or homologous recombination.
• unwinding of the helix ► The RecA protein in E. coli promotes the exchange of reciprocal single-stranded DNA
• reducing increased coiling generated during unwinding molecules (step c in the model).
• synthesis of a primer for initiation ► Other enzymes are also involved in the nicking and unwinding of DNA during
• discontinuous synthesis of the second strand recombination (RecB, RecC, and RecD).
• removal of the RNA primers ► Genetic recombination involves:
• joining of the gap-filling DNA to the adjacent strand • Alignment of two DNA duplexes or homologs (step a)
• proofreading • Endonuclease nicking (step b)
► DnaA binds to the origin of replication and is responsible for • Strand displacement (step c)
the initial steps in unwinding the helix. • Ligation [heteroduplex DNA molecules] (step d)
► Subsequent binding of DnaB and DnaC further opens and • Branch migration (step e)
destabilizes the helix. • Duplex separation (step f)
► Single-stranded binding proteins (SSBPs) stabilize the open • 180° rotation of the bottom portions [Holliday structure or
conformation. Holliday Junction] (step g)
► Proteins such as these, which require the energy normally • Endonuclease nicking (step h)
supplied by the hydrolysis of ATP to break hydrogen bonds and • Ligation & duplex separation [recombinant duplexes] (step i)
denature the double helix, are called helicases.
► Unwinding produces supercoiling that is relieved by DNA Gene Conversion Is a Consequence of DNA Recombination
gyrase, a member of a larger group of enzymes referred to as ► Gene conversion is characterize d by nonreciprocal genetic exchange
DNA topoisomerases. between two closely linked genes.
► To elongate a polynucleotide chain, DNA polymerase III requires a primer Reciprocal recombination (a X b☻)

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with a free 3'-hydroxyl group.
► The enzyme primase synthesizes an RNA primer that provides the free 3'- ab and ☻ genotypes OR
hydroxyl required by DNA polymerase III. a☻ and b genotypes
► As the replication fork moves, only one strand can serve as a template for continuous
Nonreciprocal recombination (a X b☻)
DNA synthesis (leading strand).
► The opposite lagging strand undergoes discontinuous DNA synthesis. ab and ☻ genotypes OR
► The lagging strand is synthesized as Okazaki fragments, each with an RNA primer.
a ☻ and b genotypes OR
► DNA polymerase I removes the primers on the lagging strand and the fragments are
joined by DNA ligase. a and b ☻ genotypes OR
► The b-subunit clamp prevents the core enzyme from falling off the template during DNA ► DNA synthesis at a
a and b ☻ genotypes OR
synthesis (processivity).
single replication fork
in prokaryotes involves:
• DNA polymerase III

► Both strands are synthesized concurrently by looping the lagging strand to invert the
• Single-stranded
binding proteins
(SSBPs)
• DNA gyrase
and ab☻ genotypes
physical, not biological, direction of synthesis. • DNA helicase
• Primase (RNA primers)

► DNA polymerase III functions as a dimer, with each core enzyme achieving synthesis on
one of the two strand.
► Proofreading and error correction are an integral part of DNA replication.
► All of the DNA polymerases in prokaryotes have 3' to 5' exonuclease
activity that allows proofreading.
► A number of genes involved in DNA replication have been identified by conditional mutations.
► A temperature-sensitive mutation is an example of a conditional mutation.
► It may not be expressed at a particular permissive temperature, but when mutant cells are
grown at a restrictive temperature, the mutant phenotype is expressed and can be studied.

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Eukaryotic DNA Synthesis Is Similar to Synthesis Prokaryotes, but More Complex
► In eukaryotic cells:
• there is more DNA than prokaryotic cells
• the chromosomes are linear
• the DNA is complexed with proteins
► This makes DNA replication more complex in eukaryotes than in bacteria.
► Eukaryotic chromosomes contain multiple origins of replication to allow the genome to be
replicated in a few hours.