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 mutation
• 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 x-rays
• 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
positions.
 Types of DNA Damage
• Spontaneous damage to
DNA.
– 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
DNA.
– 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
chemicals.
– 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
G.
 Types of DNA Damage

• Reaction with
carcinogens:
many carcinogens
results in the
addition of bulky
groups to the
DNA molecule
What can be done to repair the
damage?
 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
chemicals.
– 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
G.
 Direct Reversal of Base Damage

• Alkylated guanine
residues results from
exposure to alkylating
agents.
• They can transfer
methyl or ethyl groups
to DNA.
• 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
DNA.
• Damaged DNA is
recognized and removed as
free bases or as nucleotides.
• The resulting gap is filled.
• Uracil is occationally
incorporated in place of
Tymine and should be
removed.
• 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 energy.
 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
mechanism.
 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 nucleotides.
• A DNA ligase covalent binds the
fresh piece into the backbone
 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
oligonucleotide).
– 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?

GGTACGATG
CCATTCTAC
 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.
– 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
ligase.
Mismatch Repair in E.coli
Mismatch Repair in mammalian
cells
 Mismatch repair in mammalian
cells
• The old and new strands of DNA is
distinguished by a different mechanism than
methylation.
• 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
cancers).
• 140,000 cancer cases per year (10% of total cancer
cases).
• 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
growth.
• Mutations occur sporadically in somatic
cells.
• In hereditary cases, inherited germ-line
mutations predispose the individual to
cancer.
 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.
 Symptoms
• Development of the outgrowth of small
benign polyps, which eventually become
malignant.
• 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
replication.
 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
mechanism.
 Double strand breaks
• X-rays induce double strand breaks on the
chromosomes.
– 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
mistakes.
 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 damage.
• 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
activity.