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Processes involved in information
restructuring Restriction and modification
DNA repair mechanisms
Recombination Gene rearrangements
Gene amplification
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DNA METHYLATION
Occurs at the polynucleotide level, with
transfer of a methyl group from S-
adenosylmethionine
In eukaryotes, 5-methylcytosine is the
only methylated base
In prokaryotes, the major methylated
base is N6 methyl adenine Methylation in bacteria occurs at specific
sites
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Methylated bases
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DNA METHYLATION
In E.coli, methylation of A residues in the
sequence 5-----GATC-----3 is involved
in mismatch repair and it plays a central
role in controlling initiation of DNA
replication
Methylation at other sites protects DNA
cleavage by restriction enzymes
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DNA METHYLATION
In animals, methylation is foundprimarily in C residues that areimmediately 5 to G residues
Corresponding C in the complementarystrand is also methyated
In plants, the sequence is 5---CpNp--3
Methylation at a particular site isheritable (maintainance methylase)
Methylation occurs after replication
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DNA METHYLATION
Responsible for tissue-specific
inactivation of genes during development
Methylation is correlated with the arrestof expression of development-related
genes by altering the structure of
chromatin
Methylation may be involved incarcinogenesis (transition mutation)
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RESTRICTION and
MOD
IFICATIO
N
Restriction endonucleases
Nongenetic phenomena
Widespread among bacteria Restriction maps
Three types of restriction-modification
systems
Each type consists of a DNA methylase
and an endonuclease
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TYPE I
Have both enzymes in one protein
molecule
The protein molecule contains 3
subunits: nuclease, methylase,
sequence recognistion determinant
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TYPE II
Widely used in research because most
cut within the recognition sequence
Homodimers
Requires divalent cation for cleavage
but not ATP
A hemimethylated DNA is a preferred
substrate for methylase but nucleasecleaves only when the recognition sites
is unmethylated on both strands
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TYPE II
Cleavage generates a 3 hydroxyl and a
5 phosphate
Some enzymes generate a 5 over hang,
some 3 and others generate a blunt end
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TYPE III
Resemble type I
Contain both nuclease and methylase
activities in a 2 subunit enzyme
Do not require ATP
Modify just 1 strand of DNA
Cleavage site is fairly close to
recognition site
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DNAREPAIR
Defense of an organism to maintain
metabolic stability
Oxidative damage with concommitant
release of reactive oxygen species
causes the formation of DNA bases
such as 8-oxoguanine
Formation of this base contributes mostsignificantly to mutagenesis
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DNAREPAIR SYSTEMS
Direct repair
Nucleotide excision repair
Base excision repair Recombinational repair
Mismatch repair
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DNA
PHOTOPRODUCTS
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DIRECT REPAIR
PHOTOREACTIVATION
- DNA photolyase repair cyclobutanepyrimidine dimers
- 370 nm is the most effective wavelength
-the enzyme contains 2 chromphores:FADH- and 5,10-methenyltetrahydrofolateor 8-hydroxy-5-deazaflavin
-recent evidence suggests that humancells do not contain the enzyme
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DIRECT REPAIR
O6 AlkylguanineAlkyltransferase
-methylated or ethylated DNA iscomparable to UV irradiation because of
base modification-alkylation can block replication. Hence,some alkylating agents are used incancer therapy
-MNU, EMS, and MNNG are thecommon alkylating agents used in thelaboratory
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DIRECT REPAIR
O6 AlkylguanineAlkyltransferase
- bases altered are mostly purines
-O6
alkylguanine is the most mutagenicbecause of its high probability in pairing
with thymine thus, producing transition.
-the enzyme is not recyclable
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M
is
m
a
t
ch
R
ep
a
i
r
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RECOMBINATION
Maintains diversity
Any process that involves formation of
new DNA from distinct DNA molecules,
such that genetic information from each
parental DNA molecule is present in the
new molecules
4 types: homologous, site-specific,transposition and illegitimate
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HOMOLOGOUS RECOMBINATION
Recombinatorial events like meiotic
recombination, conjugation during
bacterial mating, bacterial
transformation and transduction allutilize homologous recombination
Most common way is to break and rejoin
DNA molecules
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SITE-SPECIFIC RECOMBINATION
Involves limited sequence homology
between recombining partners
Sites of breaking and joining are
determined by specific DNA-protein
interactions
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Characteristics ofDifferent Types
ofRecombination
TYPE
REQUIREMENTS
SEQUENCE
HOMOLOGY
RecA protein or
Counterpart
Sequence
specific enzyme
Homologous Yes yes No
Site-specific Yes (~ 15 bases) No Yes
Transposition No No Yes
Illegitimate no No Unknown
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GENE REARRAGEMENTS
Confers genome plasticity
Antibody diversity
Transposable elements Retroviruses
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ANTIBODYDIVERSITY
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ANTIBODYDIVERSITY
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TRANSPOSABLE ELEMENTS
Depending on whether the sequences
are oriented identically or in reverse,
homologous recombination between
them can yield deletions or inversions
Can activate or inactivate the gene into
which they move
Three classes of transposable elementsin bacteria depends on the involvement
of the transposase and resolvase
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CLASS I
Encodes a transposase but not a resolvase
Two types of class I: insertion sequenceand composite transposon
Insertion sequence is the simplesttransposable element.
It consists a gene for trasnposase flankedby 2 short inverted sequence repeats about15-25 bases pairs
Composite transposons consits of a proteincoding gene (antibiotic resistance) flankedby 2 insertion sequences
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CLASS II
Contain one set of short flanking direct
repeats
In addition to a protein-coding gene and
a transposase gene, a resolvase gene is
also present
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CLASS III
Belong to small group of bacteriophages
Phage Mu is the best studied
Inserts its chromosome at random in thehost chromosome by transpositional
mechanism and also replicate its
genome by the same mechanism
One gene encodes the transposase andanother gene encodes a protein with
DNA dependent ATPase activity
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Structures ofDifferent Classes of
Transposons
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TRANSPOSONS
Transposons and IS inserts at specific
traget sequence of 5 or 9 bp
Insertion involves duplication of that site
The two copies are present on each side
of the integrated element (action of
transposase)
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