Genetic Material-DNA

download report

Transcript Genetic Material-DNA

Genetic Material-DNA
6 November 2003
Reading:The Cell; Chapter 5,
pages: 192-201
DNA Repair
• In the living cell, DNA undergoes
frequent chemical change, especially
when it is being replicated. Most of these
changes are quickly repaired.
• A failure to repair DNA produces a
• The human genome has already revealed
130 genes whose products participate in
DNA repair.
Agents that Damage DNA
• Certain wavelengths of radiation
• ionizing radiation such as gamma rays and xrays
• ultraviolet rays, especially the UV-C rays (~260
nm) that are absorbed strongly by DNA but also
the longer-wavelength UV-B that penetrates the
ozone shield.
• Highly-reactive oxygen radicals produced
during normal cellular respiration as well as by
other biochemical pathways.
Agents that Damage DNA
• Chemicals in the Environment
• many hydrocarbons, including some found in
cigarette smoke
• some plant and microbial products
• Chemicals used in chemotherapy, especially
chemotherapy of cancers
Types of DNA Damage
• All four of the bases in DNA (A, T, C, G)
can be covalently modified at various
Types of DNA Damage
• Spontaneous damage to
– One of the most frequent is
the loss of an amino group
("deamination") - resulting,
for example, in a C being
converted to a U.
Types of DNA Damage
• Spontaneous damage to
– Depurination: cleavage of the
bond between the purine
bases and the sugar, leaving
apurinic site (AP) in DNA
Types of DNA Damage
• DNA damage
induced by
radiation and
– Formation of
pyrimidine dimers.
Types of DNA Damage
• Alkylation:
addition of methyl
or ethyl groups to
various positions on
the DNA bases.
Instead of C, T is
put to complement
Types of DNA Damage
• Reaction with
many carcinogens
results in the
addition of bulky
groups to the
DNA molecule
What can be done to repair the
DNA Repair
• Direct reversal of of the chemical
reaction that causes DNA damage
• Removal of the damaged base.
Types of DNA Damage
• DNA damage
induced by
radiation and
– Formation of
pyrimidine dimers.
Direct Reversal of Base Damage
• Pyrimidine dimers
– UV-induced damage causes
skin cancers.
– Cyclobutane ring results
from the saturation of the
double bonds between
carbons 5 and t.
– Formation of such dimers
distort DNA structure
• Photoreactivaton provides
energy to break the
cyclobutane ring. Humans
lack this mechanism.
Types of DNA Damage
• Alkylation:
addition of methyl
or ethyl groups to
various positions on
the DNA bases.
Instead of C, T is
put to complement
Direct Reversal of Base Damage
• Alkylated guanine
residues results from
exposure to alkylating
• They can transfer
methyl or ethyl groups to
• O6 -methylguanine
transferase transfers a
methyl group from DNA
to a cysteine residue in
its active site. Humans
have this mechanism.
Excision Repair
• General means to repair
• Damaged DNA is
recognized and removed
as free bases or as
• The resulting gap is
• Uracil is occationally
incorporated in place of
Tymine and should be
• Uracil can be formed by
deamination of cytosine.
Base Excision Repair
• Removal of the damaged
base. “Base excision
repair”. This is done by
a DNA glycosylase.
• Removal of its
deoxyribose phosphate
in the backbone,
producing a gap.
• Replacement with the
correct nucleotide. This
relies on DNA
polymerase ,
• Ligation of the break in
the strand with DNA
ligase. This requires ATP
to provide the needed
Nucleotide excision repair
• Widespread form of DNA repair.
• Damaged bases are removed as part of an
oligonucleotide containing the lesion.
• UV induced pyrimidine dimers and bulky
group addition can be repaired by this
Nucleotide excision repair
• The damage is
recognized by one or
more protein factors that
assemble at the location.
• Cuts are made on both
the 3' side and the 5' side
of the damaged area so
the tract containing the
damage can be removed.
• DNA synthesis - using
the intact (opposite)
strand as a template fills in the correct
• A DNA ligase covalent
binds the fresh piece into
In E.coli
• Three genes, uvrA, uvrB, uvrC.
• What happens if these genes are mutated?
– The bacteria become highly sensitive to UV (gets
damaged by it).
– UvrA-recognizes the damaged DNA and recruits UvrB
and UvrC to the damaged area.
– UvrB and UvrC then cleave the 3’ and 5’ sides of the
damaged site.
– UvrABC comples is called exinuclease (excise an
– Helicase is needed to remove the damaged area; gap is
filled with polymerase and ligase.
In eukaryotes
• RAD genes (radiation sensitivity) mutants have
increased sensitivity to UV exposure.
• Inherited diseases that result from deficiencies in
ability to repair DNA damage.
– Xeroderma pigmentosum (XP)-sensitive to UV,
develop skin cancers. They cant carry out nucleotide
excision repair.
– XPA to XPG (seven repair genes) highly homologous to
yeast RAD genes.
Mismatch Repair
• Mismatch repair deals with correcting
mismatches of the normal bases; that is,
failures to maintain normal Watson-Crick base
pairing (A.T, C.G)
• Many of the mismatched bases are removed
during replication by the proofreading activity
of DNA polymerase. Missed ones are subject to
mismatch repair!!!
• Mutations in either of these genes predisposes
the person to an inherited form of colon cancer.
(Do not forget to read the box @ page 198.
How could the mismatched base
be understood?
Mismatch repair in E. coli
• Scans newly replicated DNA, if found
enzymes of this system can identify and
repair the mismatched base from newly
replicated DNA.
• In E.coli, methylation indicates parental
strand; Adenine residues in the sequence
GATC forms 6-methyladenine. Methylation
occurs after replication.
Mismatch repair in E.coli
• MutS protein initiates repair because it
recognizes the mismatch and forms a
complex with two other proteins MutL and
– MutH is an endonuclease that can cleave the
unmethylated DNA strand.
– MutL and MutS then excise the DNA between
the strand break and gap is filled with Pol and
Mismatch Repair in E.coli
Mismatch Repair in mammalian
Mismatch repair in mammalian
• The old and new strands of DNA is
distinguished by a different mechanism than
• Presence of single strand breaks indicate
newly replicating DNA or associations
between MutS and MutL homologs also
indicate which strand is new.
Colon Cancer
• Cancers of the colon and rectum (colorectal
• 140,000 cancer cases per year (10% of total cancer
• Mostly non inherited.
• Inherited cases:
– Familial adenomatous polyposis (rare, 1%)
– Heretidary nonpolyposis colorectal cancer (15%).
Molecular Basis
• Mutated genes involved in cell
proliferation, leading to uncontrolled
• Mutations occur sporadically in somatic
• In hereditary cases, inherited germ-line
mutations predispose the individual to
The gene
• Human homology of E.coli MutS gene
involved in mismatch repair of DNA is
responsible for 50% of HNPCC.
• Three other genes also involved in repair
may be responsible.
• Defects in these genes result in high
frequency of mutations in other cells.
• Development of the outgrowth of small
benign polyps, which eventually become
• Polyps can be removed surgically. Early
diagnosis is important.
Postreplication Repair
• Recombinational
repair relies on
replacement of
damaged DNA by
recombination with an
undamaged molecule.
• Happens during
Recombinational Repair
• Normal replication is blocked with a TT dimer.
• Downstream of the damage replication goes on.
• Undamaged parental strand (which has been
replicated) is then used as a template, new strand
is synthesized based on this.
• TT dimer later is dealth with an excision repair
Double strand breaks
• X-rays induce double strand breaks on the
– Ligate the ends of the chromosomes (risky,
possible errors (loss of bases at the ends).
– Homologous recombination provides new
templates at the site of the double strand break.
Error-prone repair
• Reversal and excision repair systems act to correct
DNA damage before replication.
• Replicative DNA synthesis requires an undamaged
DNA strand as a template.
– What about the damage at the replciation fork, when TT
dimers for example block the replication.
– Cells have specialized Polymerases to replicate across a
damaged site but these polymerases lead to a lot
Error-prone polymerases
• In E. coli Polymerase V is induced in response to
UV irradiation and can synthesize a new DNA
strand across from a thymine dimer.
• E. coli Pol II and Pol IV are induced by DNA
• Characteristically error-prone DNA polymerases
exhibit low fidelity (100 to 10,000 times higher
than replicative polymerases; E.coli PolII and
eurkaryotic epsilon).
• Error prone polymerases lack 3’ 5’ proofreading