Slide 2 Molecular Biology Course SECTION F DNA damage, repair,
and recombination link Slide 3 F1F1 Mutagenesis ( F1F1 Mutagenesis
( DNA damage, repair & recombination F2F2DNA damage F2F2DNA
damage Mutation: replication fidelity, mutagens, mutagenesis F3F3
DNA repair F3F3 DNA repair DNA lesions: oxidative damage,
alkylation, bulky adducts Photoreaction, alkyltransferase, excision
repair, mismatch repair, hereditary repair defects Homologous
recombination, site-specific recombination, transposition
F4F4Recombination F4F4Recombination Slide 4 DNA damage, repair
& recombination F1F1 Mutagenesis ( F1F1 Mutagenesis ( Mutation
Replication fidelity Mutagens: chemical & physical Mutagenesis:
direct & indirect Slide 5 Mutation Replication Fidelity (
Mutagens Mutagenesis back Slide 6 F1-1 Mutation F1 Mutaagenesis
Permanent, heritable alterations in the base sequence of DNA
Reasons 1.Spontaneous errors in DNA replication or meiotic
recombination 2.A consequence of the damaging effects of physical
or chemical mutagens on DNA Slide 7 TransitionTransition : Purine
or pyrimidine is replaced by the other A GT C Transversion: a
purine is replaced by a pyrimidine or vice verseTransversion A T or
C T A or G G T or C C A or G Point mutation (a single base change)
F1 Mutaagenesis Slide 8 Noncoding DNA Nonregulatory DNA 3 rd
position of a codon Silent mutation Coding DNA altered AA Missense
mutation Phenotypic effects No Coding DNA stop codon truncated
protein Nonsense mutation Effects of a point mutation F1
Mutaagenesis Yes or No Yes Slide 9 Insertions or deletions
Frameshift mutations The ORF of a protein encoded gene is changed
so that the C-terminal side of the mutation is completely changed.
The addition or loss of one or more bases in a DNA region F1
Mutaagenesis Slide 10 Examples of deletion mutations Slide 11 F1-2
Replication fidelity F1 Mutaagenesis Mutation relevant
1.Spontaneous errors in DNA replication is very rare, one error per
10 10 base in E. coli. Important for preserve the genetic
information from one generation to the next Slide 12 Molecular
mechanisms for the replication fidelity 1.DNA polymerase:
Watson-Crick base pairing 2.3 5 proofreading exonuclease. 3.RNA
priming: proofreading the 5 end of the lagging strand 4.Mismatch
repair (F3) F1 Mutaagenesis Slide 13 by E. coli polymerase
Proofreading F1 Mutaagenesis Slide 14 F1-3&4: Mutagens F1
Mutaagenesis Mutation relevant Cause DNA damage that can be
converted to mutations. Slide 15 Physical mutagens High-energy
ionizing radiation: X-rays and g-rays strand breaks and base/sugar
destruction Nonionizing radiation : UV light pyrimidine dimers
Chemical mutagens Base analogs: direct mutagenesis Nitrous acid:
deaminates C to produce U Alkylating agents Arylating agents
Lesions-indirect mutagenesis (F2) F1 Mutaagenesis Slide 16 Base
analogs: derivatives of the normal bases incorporated in DNA,
altering base pairing properties. Nitrous acid: deaminates C to
produce U, resulting in GC AU Slide 17 F1-3&4: Mutagenesis F1
Mutaagenesis The molecular process in which the mutation is
generated. Note: the great majority of lesions introduced by
chemical and physical mutagens are repaired by one or more of the
error-free DNA repair mechanisms before the lesions is encounter by
a replication fork Slide 18 Direct mutagenesis The stable,
unrepaired base with altered base pairing properties in the DNA is
fixed to a mutation during DNA replication. F1 Mutaagenesis Slide
19 5-BrU : G : A enol form Br OH H O Br Keto form H O AGCTTCCTA
TCGAAGGAT AGCTBCCTA TCGAAGGAT 1.Base analog incorporation AGCTBCCTA
TCGAGGGAT AGCTTCCTA TCGAAGGAT 2.1st round of replication AGCTBCCTA
TCGAAGGAT AGCTCCCTA TCGAGGGAT 3.2nd round of replication AT GC
transition F1 Mutaagenesis Slide 20 Indirect mutagenesis The
mutation is introduced as a result of an error-prone repair.
Translesion DNA synthesis to maintain the DNA integrity but not the
sequence accuracy: when damage occurs immediately ahead of an
advancing fork, which is unsuitable for recombination repair (F4),
the daughter strand is synthesized regardless of the the base
identity of the damaged sites of the parental DNA. F1 Mutaagenesis
Slide 21 E. coli translession replication: SOS response: Higher
levels of DNA damage effectively inhibit DNA replication and
trigger a stress response in the cell, involving a regulated
increase (induction) in the levels of a number of proteins. This is
called the SOS response. 1.Some of the induced proteins, such as
the UvrA and UvrB proteins, have roles in normal DNA repair
pathways. 2.A number of the induced proteins, however, are part of
a specialized replication system that can REPLICATE PAST the DNA
lesions that block DNA polymerase III. back F1 Mutaagenesis Slide
22 Proper base pairing is often impossible and not strictly
required at the site of a lesion because of the SOS response
proteins, this translesion replication is error-prone. The
resulting increase in mutagenesis does not contradict the general
principle that replication accuracy is important (the resulting
mutations actually kill many cells). This is the biological price
that is paid, however, to overcome the general barrier to
replication and permit at least a few mutant cells to survive.
Slide 23 DNA damage and repair Mutagen ( Completely repaired DNA
damage (lesions) chemical reactivity of the bases Error-free
Repairing mutations Indirect mutagenesis F DNA damage, repair and
recombination minor or moderate Extensive, right before R
eplication F ork (not repairable) Direct mutagenesis Slide 24 DNA
damage, repair & recombination F2F2DNA damage F2F2DNA damage
DNA lesions: oxidative damage Alkylation bulky adducts Slide 25 DNA
lesions (DNA Oxidative damage ( Alkylation ( Bulky adducts (
1.Occurs under Normal conditionOccurs under Normal condition
2.Increased byIncreased by ionizing radiation (physical mutagens)
Alkylating agents (Chemical mutagens) UV light (physical mutagens)
Carcinogen (Chemical mutagens) DNA damage, repair &
recombination Slide 26 F2-1DNA lessions An alteration to the normal
chemical or physical structure of the DNA Slide 27 The biological
effect of the unrepaired DNA lesions Lethal (cell death) Physical
distortion of the local DNA structure Blocks replication And/or
transcription Mutagenic Allowed to Remained in the DNA A mutation
could become fixed by direct or indirect mutagenesis Living cell
Altered chemistry of the bases DNA lessions Slide 28 Spontaneous
DNA lesions 1.Inherent chemical reactivity of the DNA 2.The
presence of normal, reactive chemical species within the cell
1.Deamination ( : C UC U methylcytosine T, hard to be detected
2.Depurination ( ) : break of the glycosylic bond, non-coding
lesion. 3.Depyrimidine ( ) back Slide 29 Chemical reactivity of
bases is responsible for some DNA lesion DNA lessions Slide 30
deamination --ATGCTACG-- --TACGATGC-- --ATGUTACG-- --TACGATGC--
--ATG TACG-- --TACGATGC-- U --ATGCTACG-- --TACGATGC-- Uracil DNA
glycosylase Cytosine deamination and repair back Slide 31 DNA
damage, repair & recombination F2-2Oxidative damage DNA lesions
caused by reactive oxygen species such as superoxide and hydroxyl
radicals Slide 32 Oxidation products 1. occurs under NORMAL
conditions in all aerobic cells due to the presence of reactive
oxygen species (ROS), such as superoxide, hydrogen peroxide, and
the hydroxyl radicals (OH). 2.The level of this damage can be
INCREEASED by hydroxyl radicals from the radiolysis of H 2 O caused
by ionizing radiation DNA damage Slide 33 DNA damage, repair &
recombination F2-3Alkylation Nucleotide modification caused by
electrophilic alkylating agents such as methylmethane sulfonate and
ethylnitrosourea ( ) Slide 34 Alkylated bases 1.Electrophilic
chemicals adds alkyl groups to various positions on nucleic acids
2.Distinct from those methylated by normal methylating enzymes.
alkylating agents Slide 35 DNA damage, repair & recombination
F2-4Bulky adducts DNA lesions that distort the double helix and
cause localized denaturation, for example pyrimidine dimers and
arylating agents adducts These lesions disrupt the normal function
of the DNA Slide 36 Cyclobutane pyrimidine dimer ( ) Guanine adduct
of benzo[a]pyrene Aromatic arylating agents Covalent adducts back
DNA damage Slide 37 DNA damage, repair & recombination F3F3DNA
repair F3F3DNA repair Photoreactivation ( ) Alkyltransferase ( )
Exision repair ( ) Mismatch repair ( ) Hereditary repair defects (
) Slide 38 DNA damage, repair & recombination F3-1:
Photoreactivation Monomerization of cyclobutane pyrimidine dimers
by DNA photolyases in the presence of visible light Direct reversal
of a lesion and is error-free Slide 39 DNA damage, repair &
recombination F3-2: Alkyltransferase Direct reversal of a lesion
and is error-free Removes the alkyl group from mutagenic O 6 -
alkylguanine which can base-pair with T. The alkyl group is
transferred to the protein itself and inactivate it. Slide 40 The
response is adaptive because it is induced in E. coli by low levels
of alkylating agents and gives increased protection against the
lethal and mutagenic effects of the high doses DNA repair Slide 41
DNA damage, repair & recombination F3-3: Excision repair
1.Includs nucleotide excision repair (NER) and base excision repair
(BER). 2.Is a ubiquitous mechanism repairing a variety of lesions.
3.Error-free repair Slide 42 Nucleotide excision repair DNA repair
1.An endonuclease cleaves DNA a precise number of bases on both
sides of the lesions (UvrABC endonulcease removes pyrimidine
dimers) 2.Excised lesion-DNA fragment is removed 3.The gap is
filled by DNA polymerase I and sealed by ligase Slide 43 Base
excision repair DNA glycolases cleaves apurinic or pyrimidine site
DNA polymerase DNA ligase DNA repair cleaves N-glycosylic bond AP
endonuclease 3 5 cleavage and & 5 3 synthesis Slide 44 DNA
damage, repair & recombination F3-3: Mismatch repair A
specialized form of excision repair which deals with any base
mispairs produced during replication and which have escaped
proofreading error-free Slide 45 The parental strand is methylated
at N 6 position of all As in GATC sites, but methylation of the
daughter strand lag a few minutes after replication MutH/MutS
recognize the mismatched base pair and the nearby GATC DNA helicase
II, SSB, exonuclease I remove the DNA fragment including the
mismatch DNA polymerase III & DNA ligase fill in the gap
Expensive to keep the accuracy back Slide 46 DNA damage, repair
& recombination Homologous recombination Site-specific
recombination Transposition F4F4Recombination F4F4Recombination
Mutation Relevance An important reason for variable DNA sequences
among different populations of the same species Slide 47 F4-1
Homologous recombination ( Diploid eukaryotes: crossing over
Haploid prokaryotes: recA-dependent, Holliday model DNA repair in
replication fork DNA damage, repair & recombination The
exchange of homologous regions between two DNA moleculs Slide 48
Diploid eukaryotesDiploid eukaryotes: crossing over 1.Homologous
chromosomes line up in meiosis (when) 2.The nonsister chromatids
exchange equivalent sections (what) F4 Recombination Slide 49
Haploid prokaryotesHaploid prokaryotes recombination Between the
two homologous DNA duplex (where) 1. between the replicated
portions of a partially duplicated DNA 2.between the chromosomal
DNA and acquired foreign DNA Holliday model (How) F4 Recombination
Slide 50 2.Nicks made near Chi (GCTGGTGG) sites by a nuclease. 3.
ssDNA carrying the 5 ends of the nicks is coated by RecA to form
RecA-ssDNA dilaments. recA-dependent bacterial homologous
recombination F4 Recombination 1.Homologous DNA pairs 35 35 3 5
Slide 51 3.RecA-ssDNA filaments search the opposite DNA duplex for
corresponding sequence (invasion). 4.form a four-branched Holliday
structurefour-branched Holliday structure 5.Branch migration back
F4 Recombination Slide 52 6.Resolving Holliday junctionesolving
Holliday junction F4 Recombination Slide 53 Slide 54 RuvAB is an
asymmetric complex that promotes branch migration of a Holliday
junction. F4 Recombination Slide 55 Recombination based DNA repair
at replication fork a.Replication encounter a DNA lesion b.Skip the
lesion & re- initiate on the side of the lesion c.Fill the
daughter strand gap by replacing it with the corresponding section
from the parental sister strand d.post-replication repair of the
left lesion Slide 56 F4-2: Site-specific recombination ( DNA
damage, repair & recombination 1.Exchange of non-homologous but
specific pieces of DNA (what) 2.Mediated by proteins that recognize
specific DNA sequences. (how) Slide 57 1.l-encoded integrase (Int):
makes staggered cuts in the specific sites 2.Int and IHF
(integration host factor encoded by bacteria): recombination and
insertion 3.l-encoded excisionase (XIS): excision of the phage DNA
Site-specific recombination: bacteriophage insertion Site-specific
recombination: bacteriophage insertion F4 Recombination Slide 58
Site-specific recombination: Antibody diversity Site-specific
recombination: Antibody diversity H and L are all encoded by three
gene segments: V, D, J VDJ Two heavy chains (H) 250155 Two light
chains (L) 2504 Enormous number (>10 8 ) of different H and L
gene sequences can be produced by such a recombination F4
Recombination Slide 59 F4-3 Transposition ( back DNA damage, repair
& recombination 1.Requires no homology between sequences nor
site-specific 2.Relatively inefficient 3.Require Transposase
encoded by the transposon ( Slide 60 Various transposons: In E.
coli: IS elements/insertion sequence, 1-2 kb, comprise a
transposase gene flanked by a short inverted terminal repeats Tn
transposon series carry transposition elements and b-lactamase
(penicillin resistance) Eukaryotic transposons, many are
retrotransposons: Yeast Ty element encodes protein similar to RT
(reverse transcriptase) F4 Recombination Slide 61 Simplified
Transposition process F4 Recombination
