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

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


1
Replication, Maintenance, and Rearrangements of
Genomic DNADNA ReplicationDNA
RepairRecombinationDNA Rearrangments
2
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.

3
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
4
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
5
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
6
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
7
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.

8
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.

9
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
10
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
11
6.10 Model of the E. coli replication fork
  • A Detailed Overview of the Replication in E.coli

12
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
13
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
14
DNA Repair
  • Telomeres
  • DNA Damage
  • Mechanisms of DNA Repair

15
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
16
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
17
6.18 Examples of DNA damage induced by radiation
and chemicals
18
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
19
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
20
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
21
6.23 Nucleotide-excision repair in mammalian
cells
  • Transcription-coupled repair is specifically
    dedicated to repairing damage within actively
    transcribed genes.

22
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
23
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
24
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
25
6.27 Repair of double strand breaks
26
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.

27
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
28
6.29 The Holliday model for homologous
recombination
  • The Holliday model is a molecular model of
    genetic recombination involving the formation of
    heteroduplex regions.

29
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

30
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
31
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.

32
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
33
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
34
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
35
DNA Amplification
  • Additional copies of genes can result from the
    replication process.
  • Gene amplification occurs as an abnormal event in
    cancer cells
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