Objectives of DNA recombination

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Objectives of DNA recombination

• The different processes of DNA recombination
  homologous recombination, site-specific
  recombination, transposition, illegitimate
  recombination, etc.
• What are the differences between these process
  (i) the DNA substrates, (ii) the enzymes used,
  and (iii) the recombinant products produced.
• General mechanism of recombination (I)
  presynapsis (initiation), (ii) synapsis (the
  formation of joint molecules), and (iii)
  postsynapsis (resolution).
• In addition to provide genetic diversity, DNA
  recombination plays an important role in repair
  of DNA double-strand breaks and DSG (to be
  discussed in the section of DNA repair).

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Examples of recombination
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Homologous recombination

• Refer to recombination between homologous DNA
  sequence in the same or different DNA molecules.
• The enzymes involved in this process can catalyze
  recombination between any pair of homologous
  sequences, as long as the size of homologous
  sequence is longer than 45 nt or longer. No
  particular sequence is required.
• Models of homologous recombination.
• Homologous recombination of E. coli.
• Meiotic recombination.

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The Holliday model of recombination
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Homologous recombination of E. coli

• Identification of genes involved in
  recombination (i) isolation of mutants affecting
  recombination in wild-type cells (eg., recA,
  recB, recC etc.), (ii) the recombinational
  deficiency in recBC cells may be suppressed by
  sbcA or sbcB mutations. The sbcB gene encodes for
  a 3 to 5 ss-DNA exonuclease, while the sbcA
  mutation activate the expression of recE which
  encodes for 5 to 3 exonuclease. (iii) isolation
  of mutants affecting recombination in recB recC
  sbcB or recB recC sbcA cells (eg., recF, recO,
  recR, recQ, recJ etc.)
• The biochemical functions of rec genes.

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Homologous recombination is catalyzed by enzymes

• The most well characterized recombination enzymes
  are derived from studies with E. coli cells.
• Presynapsis helicase and/or nuclease to generate
  single-strand DNA with 3-OH end (RecBCD) which
  may be coated by RecA and Ssb.
• Synapsis joint molecule formation to generate
  Holliday juncture (RecA).
• Postsynapsis branch migration and resolution of
  Holliday juncture (RuvABC).

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RecBCD

• A multifunctional protein that consists of three
  polypeptides RecB (133 kDa), RecC (129 kDa) and
  RecD (67 kDa).
• Contain nuclease (exonuclease and Chi-specific
  endonuclease) and helicase activity.

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Chi-specific nicking by RecBCD
5-GCTGGTGG-3
Fig. 22.7
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Helicase and nuclease activities of the RecBCD
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The Bacterial RecBCD System Is Stimulated by chi
Sequences
FIGURE 15.17 RecBCD unwinding and cleavage
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The RecBCD pathway of recombination
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RecA binds selectively to single-stranded DNA
Fig. 22.4
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RecA forms nucleoprotein filament on
single-strand DNA
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Fig. 22.5
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Paranemic joining of two DNA (in contrast to
plectonemic)
Fig. 22.6
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Strand-Transfer Proteins Catalyze Single-Strand
Assimilation

• RecA forms filaments with single-stranded DNA and
  catalyzes the assimilation of single-stranded
  DNA to displace its counterpart in a DNA duplex.

FIGURE 15.18 RecA strand invasion
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RuvABC

• RuvA (22 kDa) binds a Holliday junction with high
  affinity, and together with RuvB (37 kDa)
  promotes ATP-dependent branch migration of the
  junctions leading to the formation of
  heteroduplex DNA.
• RuvC (19 kDa) resolves Holliday juncture into
  recombinant products.

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Fig. 22.9
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Fig. 22.10
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Fig. 22.14
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Fig. 22.15
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Fig. 22.17
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Homologous Recombination Occurs between Synapsed
Chromosomes in Meiosis

• Chromosomes must synapse (pair) in order for
  chiasmata to form where crossing-over occurs.
• The stages of meiosis can be correlated with the
  molecular events at the DNA level.

FIGURE 03 Recombination occurs at specific
stages of meiosis
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Fig. 15.13
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Fig. 15.15
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The Synaptonemal Complex Forms after
Double-Strand Breaks

• Double-strand breaks that initiate recombination
  occur before the synaptonemal complex forms.
• If recombination is blocked, the synaptonemal
  complex cannot form.
• Meiotic recombination involves two phases one
  that results in gene conversion without
  crossover, and one that results in crossover
  products.

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Fig. 22.18
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Fig. 22.19
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Fig. 22.20
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Fig. 22.21
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Fig. 22.24
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Gene conversion the phenomenon that abnormal
ratios of a pair of parental alleles is detected
in the meiotic products. At the molecular level
the conversion of one genes sequence to that of
another.
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Fig. 22.25
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Fig. 22.26
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Site-specific Recombination Bacteriophage lambda
integration in E. coli
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Fig. 15.28
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A site-specific recombination reaction (eg.
catalyzed by Int of bacteriophage lambda)
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Fig. 15.31
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Recombination Pathways Adapted for Experimental
Systems
FIGURE 15.38 Cre/lox system for gene knockouts
Adapted from H. Lodish, et al. Molecular Cell
Biology, Fifth edition. W. H. Freeman Company,
2003.
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Fig. 23.21
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Fig. 23.12
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Fig. 23.13
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Fig. 23.14
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Fig. 23.15
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Fig. 23.16
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Fig. 23.17
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Transposition

• Transposition is mediated by transposable
  elements, or transposons.
• Transposons of bacteria IS (insertion sequences)
  contains only sequences required for
  transposition and proteins (transposases) that
  promote the process. Complex transposons contain
  genes in addition to those needed for
  transposition.
• Transposition is characterized by duplication of
  direct repeats (5-9 bps in most cases) at target
  site.
• Transposition, in some instances, may be mediated
  through a RNA intermediate (retrotransposons and
  non-LTR retrotransposons).

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Duplication of the DNA sequence at a target site
when a transposon is inserted
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Fig. 23.1
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Fig. 23.2
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Fig. 17.3
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Fig. 23.3
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Fig. 23.4
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Fig. 23.5
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Replicative transposition is meidated by a
cointegrate intermediate.
Fig. 23.6
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Fig. 23.7
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Eukaryotic transposons

• DNA transposons (i) Ds and Ac of maize, (ii)
  Drosophila P elements.
• Retrotransposons (i) LTR retrotransposons (Ty
  element of yeast and copia of Drosophila). (ii)
  non-LTR retrotransposons (LINES, Alu, group II
  introns).

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Ds and Ac of maize
Fig. 23.8
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Fig. 23.9
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Fig. 23.10
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Hybrid Dysgenesis
Fig. 17.20
F
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Fig. 17.21
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Fig. 17.22
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Fig.23.19
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Fig. 23.18
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Fig. 23.20
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Fig. 23.21
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Fig. 23.22
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Fig. 23.23
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Fig. 23.24
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Nonviral transposons LINES
Fig. 23.25
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Fig. 23.26
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Fig. 23.27
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Fig. 23.28
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Group II introns Retrohoming
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DNA Repair

• DNA damage may arise (i) spontaneously, (ii)
  environmental exposure to mutagens, or (iii)
  cellular metabolism.
• DNA damage may be classified as (I) strand
  breaks, (ii) base loss (AP site), (iii) base
  damages, (iv) adducts, (v) cross-links, (vi)
  sugar damages, (vii) DNA-protein cross links.
• DNA damage, if not repaired, may affect
  replication and transcription, leading to
  mutation or cell death.

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Fig. 20.27
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Fig. 20.28
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Fig. 20.29
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Methylataion and Mismatch Repair
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Model for Mismatch Repair
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Base-Excision Repair
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FIGURE 16.12 Uracil is removed from DNA
FIGURE 16.13 Glycosylases remove bases
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16.5 Base Excision Repair Systems Require
Glycosylases
FIGURE 16.14 Base removal triggers excision
repair
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Nucleotide-Excision Repair in E. coli and Humans
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Alkylation of DNA by alkylating agents
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Direct Repair Photoreactivation by photolyase
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O6-methyl G, if not repaired, may produce a
mutation
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Direct Repair Reversal of O6 methyl G to G by
methyltransferase
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Direct repair of alkylated bases by AlkB.
Direct re
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Effect of DNA damage on replication (i) coding
lesions wont interfere with replication but may
produce mutation, (ii) non-coding lesions will
interfere with replication and may lead to
formation of daughter-strand gaps (DSG) or
double-strand breaks (DSB).
DSG and DSB may be repaired by recombination
process, to be discussed in the following section.
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Models for recombinational DNA repair
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Fig. 20.40
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Fig. 20.38
Model for nonhomologous end-joining
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Figure 16.25 NHEJ requires several reactions.
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Fig. 20.41