Replication, Maintenance, and Rearrangements of Genomic DNA DNA Replication DNA Repair Recombination DNA Rearrangments

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Replication, Maintenance, and Rearrangements of
Genomic DNADNA ReplicationDNA
RepairRecombinationDNA Rearrangments
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Introduction

• In order for species to evolve, mutations and
  gene rearrangements are needed to maintain
  genetic variation between individuals.
• DNA replication is much more complex than a
  single enzymatic reaction other proteins and
  specific DNA sequences are also involved.
• Proofreading mechanisms are required to ensure
  that the accuracy of replication is compatible
  with the low frequency of errors that is needed
  for cell reproduction.

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DNA Polymerases

• DNA polymerase catalyzes the synthesis of DNA.
• Cells have multiple different DNA polymerases.
• All polymerases synthesize DNA only in the 5 to
  3 direction.
• DNA polymerases add new deoxyribonucleotides only
  to primer strands that are hydrogen-bonded to the
  parental DNA.

Figure 6.1 The reaction catalyzed by DNA
polymerase
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The Replication Fork

• Growing E. coli in the presence of radioactive
  thymidine 3H initially allowed subsequent
  visualization of newly replicated DNA by
  autoradiography.
• A replication fork is the region of DNA synthesis
  where the parental strands separate and two new
  daughter strands elongate.
• Only one strand of DNA is synthesized in a
  continuous manner in the direction of overall DNA
  replication.

Figure 6.2 Replication of E. coli DNA
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The Replication Fork

• The leading strand is the strand of DNA that is
  synthesized continuously in the direction of
  movement of the replication fork.
• The lagging strand is the strand of DNA
  synthesized opposite to the direction of movement
  of the replication fork, by ligation of Okazaki
  fragments.
• Okazaki fragments are small pieces of newly
  synthesized DNA that are joined to form an intact
  new DNA strand.

Figure 6.3 Synthesis of leading and lagging
strands of DNA
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Initiation of Lagging Strand Synthesis

• Primase is used to synthesized RNA primers.
• DNA polymerase synthesizes Okazaki fragments.
• DNA Ligase glues the Okazaki fragments together.

Figure 6.4 Initiation of Okazaki fragments with
RNA primers
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Lagging Strand Synthesis

• Primase is an enzyme that synthesizes short
  fragments of RNA complementary to the lagging
  strand template at the replication fork.
• An exonuclease is an enzyme that hydrolyzes DNA
  molecules in either the 5 to 3 or 3 to 5
  direction.
• RNase H is an enzyme that degrades the RNA strand
  of RNA-DNA hybrids, and 5 to 3 exonucleases.

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Polymerase and Replication
Figure 6.7 Polymerase accessory proteins

• One class of proteins required for replication
  binds to DNA polymerases, increasing the activity
  of the polymerases and causing them to remain
  bound to the template DNA so that they continue
  synthesis of a new DNA strand.
• Other proteins unwind the template DNA and
  stabilize single-stranded regions.

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The Replication Fork

• Helicases are enzymes that catalyze the unwinding
  of parental DNA.
• Single-stranded DNA-binding proteins stabilize
  the unwound template DNA.
• As the strands of parental DNA unwind, the DNA
  ahead of the replication fork is forced to rotate.

Figure 6.8 Action of helicases and
single-stranded DNA-binding proteins
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The Replication Fork

• Topoisomerases are enzymes that catalyze the
  reversible breakage and rejoining of DNA strands.
• The enzymes involved in DNA replication act in a
  coordinated manner to synthesize both leading and
  lagging strands of DNA simultaneously at the
  replication fork.

Figure 6.9 Action of topoisomerases during DNA
replication
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6.10 Model of the E. coli replication fork

• A Detailed Overview of the Replication in E.coli

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Origins and the Initiation of Replication

• Origins of replication serve as binding sites for
  proteins that initiate the replication process.
• Single origins are sufficient to direct the
  replication of bacterial and viral genomes, but
  multiple origins are needed to replicate the much
  larger genomes of eukaryotic cells within a
  reasonable period of time.

Figure 6.12 Origin of replication in E. coli
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The Fidelity of Replication

• The accuracy of DNA replication is critical to
  cell reproduction.
• One mechanism by which DNA polymerase increases
  the fidelity of replication is by helping to
  select the correct base for insertion into newly
  synthesized DNA.
• Proofreading is the selective removal of
  mismatched bases by DNA polymerase.
• DNA polymerases require primers and catalyze the
  growth of DNA strands only in the 5 to 3
  direction.

Figure 6.11 Proofreading by DNA polymerase
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DNA Repair

• Telomeres
• DNA Damage
• Mechanisms of DNA Repair

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Telomeres and Telomerase Maintaining the Ends of
Chromosomes

• Telomeres are repeats of simple-sequence DNA that
  maintain the ends of linear chromosomes.
• A reverse transcriptase is a DNA polymerase that
  uses an RNA template.
• Defects in telomerase and the normal maintenance
  of telomeres are associated with several human
  diseases.

Figure 6.16 Action of telomerase
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DNA Repair

• Mutations in DNA can result from the
  incorporation of incorrect bases during DNA
  replication.
• They may be
• Spontaneous
• Induced by exposure to chemicals or radiation.

Figure 6.17 Spontaneous damage to DNA
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6.18 Examples of DNA damage induced by radiation
and chemicals
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Direct Reversal of DNA Damage

• Pyrimidine dimers are a common form of DNA damage
  caused by UV light in which adjacent pyrimidines
  are joined to form a dimer.
• Photoreactivation is a process where energy
  derived from visible light is utilized to break
  the cyclobutane ring structure.
• Another form of direct repair deals with damage
  resulting from the reaction between alkylating
  agents and DNA.

Figure 6.19 Direct repair of thymine dimers
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Base Excision Repair

• Base-excision repair is a process in which single
  damaged bases are recognized and removed from the
  DNA molecule.
• DNA glycosylase cleaves the bond linking the base
  (uracil) to the deoxyribose of the DNA backbone.
• AP endonuclease cleaves adjacent to AP sites in
  DNA.

Figure 6.21 Base-excision repair
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Enzymes Involved in Excision Repair

• Nucleotide-excision repair is a mechanism of DNA
  repair in which oligonucleotides containing
  damaged bases are removed from a DNA molecule.
• Excinuclease is the protein complex that excises
  damaged DNA during nucleotide-excision repair in
  bacteria.

Figure 6.22 Nucleotide-excision repair of
thymine dimers
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6.23 Nucleotide-excision repair in mammalian
cells

• Transcription-coupled repair is specifically
  dedicated to repairing damage within actively
  transcribed genes.

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Mismatch Excision Repair

• The mismatch repair system scans newly replicated
  DNA and identifies and excises mismatched bases.
• DNA of E. coli is modified by the methylation of
  adenine residues with the sequence GATC to form
  6-methyladenine.

Figure 6.24 Mismatch repair in E. coli
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Translesion DNA Synthesis

• Translesion DNA synthesis provides a mechanism by
  which the cell can bypass DNA damage at the
  replication fork, which can then be corrected
  after replication is complete.
• The enzyme polymerase V is induced in response to
  extensive UVA irradiation and can synthesize a
  new DNA strand across from a thymine dimer.

Figure 6.25 Translesion DNA synthesis
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Recombinational Repair

• Recombinational repair is a means of DNA repair
  that relies on replacement of the damaged DNA by
  recombination with an undamaged molecule.
• It provides a major mechanism for repair of
  double strand breaks.

Figure 6.26 Recombinational repair
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6.27 Repair of double strand breaks
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Recombination between Homologous DNA Sequences

• Recombination is key to the generation of genetic
  diversity, which is critical from the standpoint
  of evolution.
• Homologous recombination is a molecular mechanism
  that involves the exchange of information between
  DNA molecules that share sequence homology over
  hundreds of bases.

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Models of Homologous Recombination

• During recombination between homologous DNA
  molecules, alignment is provided by complementary
  base pairing strands.
• Homologous recombination leads to the formation
  of heteroduplex regions.

Figure 6.28 Homologous recombination by
complementary base pairing
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6.29 The Holliday model for homologous
recombination

• The Holliday model is a molecular model of
  genetic recombination involving the formation of
  heteroduplex regions.

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DNA Rearrangements

• Several types of DNA rearrangements are now
  recognized in both prokaryotic and eukaryotic
  cells.
• Transposable elements constitute a large fraction
  of the genomes of plants and animals, including
  nearly half of the human genome.
• Site-specific recombination is mediated by
  proteins that recognize specific DNA sequences,
  such as antibodies or cell receptors

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DNA Rearrangements

• Immunoglobulins consist of pairs of identical
  heavy and light polypeptide chains.
• The genes that encode immunoglobulin light chains
  consist of three regions a V region, a joining
  (J) region, and a C region.
• Heavy-chain genes include a fourth region known
  as the diversity, or D, region, which encodes
  amino acids lying between V and J.

Figure 6.36 Structure of an immunoglobulin
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6.38 Rearrangement of immunoglobulin heavy-chain
genes

• DNA rearrangements are initiated by introducing a
  double strand break between the recombination
  signal sequences and the coding sequences.

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Site-Specific Recombination

• Class switch recombination is a type of
  region-specific recombination responsible for the
  association of rearranged immunoglobulin V(D)J
  regions with different heavy chain constant
  regions.
• Class switch recombination transfers a rearranged
  variable region to a new downstream constant
  region, with deletion of the intervening DNA.

Figure 6.41 Class switch recombination
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Transposition via DNA Intermediates

• Transposable elements, or transposons, are DNA
  sequences that can move to different positions in
  the genome.
• The first transposons that were characterized in
  bacteria, which move via DNA intermediates.

Figure 6.43 Bacterial transposons
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Transposition via RNA Intermediates

• Retrotransposons are transposable elements that
  move via reverse transcription of an RNA
  intermediate.
• Retroviruses contain RNA genomes in their virus
  particles but replicate via the synthesis of a
  DNA provirus.
• Reverse transcriptase is a DNA polymerase that
  uses an RNA template.

Figure 6.44 The organization of retroviral DNA
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DNA Amplification

• Additional copies of genes can result from the
  replication process.
• Gene amplification occurs as an abnormal event in
  cancer cells