Lecture 4: Errors, Repair, SNP Flashcards
Types of DNA damage
- Single strand breaks
- Double strand breaks
- Pyrimidine Dimer
- Nucleotide base oxidation
Cause of DNA damage
Exogenous
Thermal disruption
UV light exposure
Ionizing radiation
Exposure to mutagens, carcinogens, and viruses
Cause of DNA damage
Endogenous
Cellular metabolism
Hydrolysis
Nuclease Digestion
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.
AGENTS THAT DAMAGE DNA
Chemicals in the environment
- many hydrocarbons, including some found in cigarette smoke
- some plant and microbial products, e.g. the aflatoxins produced in moldy peanuts
- Chemicals used in chemotherapy, especially chemotherapy of cancers
AGENTS THAT DAMAGE DNA
Intrinsic Spontaneous mutation
- most error during DNA replication by error of polymerase 3’ to 5’ exonuclease activity
- MMR enzyme mutation caused mismatch repair failure
- Highly-reactive oxygen radicals produced during normal cellular respiration as well as
by other biochemical pathways.
REPAIRING DAMAGED BASES
The recent publication of the human genome has revealed _________ genes whose
products participate in DNA repair. More is expected to be discovered
130
REPAIRING DAMAGED BASES
- Damaged or inappropriate bases can be repaired by several mechanisms:
- Direct chemical reversal of the damage
- Excision Repair, in which the damaged base or bases are removed and then replaced with
the correct ones in a localized burst of DNA synthesis. There are three modes of excision
repair, each of which employs specialized sets of enzymes. - Base Excision Repair (BER)
- Nucleotide Excision Repair (NER)
- Mismatch Repair (MMR)
REPAIRING DAMAGED BASES
Excision Repair
In which the damaged base or bases are removed and then replaced with
the correct ones in a localized burst of DNA synthesis. There are three modes of excision
repair, each of which employs specialized sets of enzymes.
REPAIRING DAMAGED BASES
The 2015 Nobel Prize in chemistry was shared by three researchers for their
pioneering work in DNA repair: Tomas Lindahl (BER), Aziz Sancar (NER), and
Paul Modrich (MMR).
DIRECT REPAIR GENES
DNA photolyase
- Natural repair system for pyrimidine dimers caused by UV damage
- Directly reverse cyclobutane pyrimidine dimer (CPD) via photochemical reactions
DIRECT REPAIR GENES
O6-methylguanine-DNA methyltransferase (MGMT)
- naturally occurring mutagenic DNA lesion O6-methylguanine back to guanine
- prevents mismatch and errors during DNA replication and transcription
- the methylation state of the MGMT gene promotor determined whether tumor cells
would be responsive to temozolomide drug therapy
BASE EXCISION REPAIR (BER)
The damaged base estimated to occur some _______ times a day in each cell in
our body!
20,000
BASE EXCISION REPAIR (BER)
Remove it by a __________. There are at least 8 genes encoding different
DNA glycosylases:
Remove it by a DNA glycosylase. There are at least 8 genes encoding different
DNA glycosylases
BASE EXCISION REPAIR (BER)
There are at least 8 genes encoding different
DNA glycosylases.
- Each enzyme responsible for identifying and removing a specific kind of base damage.
- Two genes encoding enzymes (AP endonuclease and DNA Exonuclease) function to
removal deoxyribose phosphate in the backbone, producing a gap
BASE EXCISION REPAIR (BER)
Replacement with the correct nucleotide. This relies on DNA polymerase _________, one of at least _____ DNA polymerases encoded by our genes
Replacement with the correct nucleotide. This relies on DNA polymerase
beta, one of at least 11 DNA polymerases encoded by our genes
BASE EXCISION REPAIR (BER)
______ of the break in the strand. Two enzymes are known that can do this;
both require ______ to provide the needed energy.
Ligation of the break in the strand. Two enzymes are known that can do this;
both require ATP to provide the needed energy.
BASE EXCISION REPAIR
One stand of DNA contains __________
base, such as _________
One stand of DNA contains deaminated
base, such as Uracil
BASE EXCISION REPAIR
DNA glycosylases scans _______
the DNA and
removes Uracil, leaving AP site
BASE EXCISION REPAIR
AP endonuclease locates _________
AP site and nicks backbone
BASE EXCISION REPAIR
DNA Exonuclease removes
nucleotides near the nick, leaving gap
BASE EXCISION REPAIR
DNA Polymerase synthesizes to _______
fill in gap
BASE EXCISION REPAIR
DNA Ligase seals the
backbone
NUCLEOTIDE EXCISION REPAIR (NER)
NER differs from BER in several ways:
- It uses different enzymes (XP products).
- Even “though there may be only a single “bad” base to correct, NER removes a large “patch around
the damage such as to removes DNA damage induced by ultraviolet light (UV), such as thymine dimer.
NUCLEOTIDE EXCISION REPAIR (NER)
The steps and some key players:
The damage is recognized by ______________, also functions in normal transcription) and
may be more protein factors that assemble at the location.
Transcription Factor IIH, (TFIIH)
NUCLEOTIDE EXCISION REPAIR (NER)
The steps and some key players:
The DNA is unwound producing a “bubble”. The enzyme system (Numerous proteins, including _____
products (____________), make cut both the ___’ side and the _____’ side of the damaged area so the
tract containing the damage can be removed.
XP ( XPA, XPB, XPF, XPG)
3’
5’
NUCLEOTIDE EXCISION REPAIR (NER)
The steps and some key players:
A fresh burst of DNA synthesis — using the intact (opposite) strand as a template — fills in the correct
nucleotides with ___________
A fresh burst of DNA synthesis — using the intact (opposite) strand as a template — fills in the correct
nucleotides with DNA polymerase delta and epsilon
XERODERMA PIGMENTOSUM (XP)
Type of disease
A rare inherited autosomal recessive
disease of humans in which a deficiency of
excinuclease occurs
XERODERMA PIGMENTOSUM (XP)
XP can be caused by
XP can be caused by mutations in any one
of several genes (XPA, XPB, XPF, XPG), all
of which have roles to play in NER
XP resulting in
- XP resulting in skin discolouration and
multiple tumours on exposure to UV light.
XP unprepared __________ in humans may lead to ___________
- Unrepaired pyrimidine dimers in humans
may lead to melanoma
MISMATCH REPAIR (MMR)
- DNA mismatch repair is a system for recognizing and repairing erroneous insertion,
deletion, and mis-incorporation of bases that can arise during ____________________, as well as repairing some forms of ____________
- DNA mismatch repair is a system for recognizing and repairing erroneous insertion,
deletion, and mis-incorporation of bases that can arise during DNA replication and
recombination, as well as repairing some forms of DNA damage
MISMATCH REPAIR (MMR)
Mismatch repair deals with correcting mismatches of the __________; that is, failures to
maintain normal ________ base pairing (AT, CG)
Mismatch repair deals with correcting mismatches of the normal bases; that is, failures to
maintain normal Watson-Crick base pairing (AT, CG)
MISMATCH REPAIR (MMR)
It can enlist the aid of enzymes involved in both ______________ and ____________ as well as using enzymes specialized for this function.
It can enlist the aid of enzymes involved in both base-excision repair (BER) and nucleotide-
excision repair (NER) as well as using enzymes specialized for this function.
MISMATCH REPAIR (MMR)
- Recognition of a mismatch requires several different proteins including one encoded by
__________ known as ________________, a caretaker gene
- Recognition of a mismatch requires several different proteins including one encoded by
MSH2 known as MutS protein homolog 2, a caretaker gene
MISMATCH REPAIR (MMR)
Cutting the mismatch out also requires several proteins, including one encoded by ______
known as ______ homologs. It forms a complex with _____ and _______, increasing the _______
footprint on the DNA.
Cutting the mismatch out also requires several proteins, including one encoded by MLH1
known as MutL homologs. It forms a complex with MutS and MutH, increasing the MutS
footprint on the DNA.
MISMATCH REPAIR (MMR)
Mutations in either of these genes predisposes the person to an inherited form of colon
cancer. So these genes qualify as _____________
Tumor suppressor genes
Mismatch Repair in Prokaryotes and Eukaryotes
Mut S recognizes
and binds mismatch
Mismatch Repair in Prokaryotes and Eukaryotes
Mut L links
S to H
Mismatch Repair in Prokaryotes and Eukaryotes
Mut H recognizes the
CH3-parental strand and makes nick on daughter strand
Mismatch Repair in Prokaryotes and Eukaryotes
In human: MutS =
hMSH (1-6); MutL = hMLH1 and hPMS2; MutH = GTBP
REPAIRING STRAND BREAKS
Single-Strand Breaks (SSBs)
- Breaks in a single strand of the DNA molecule are repaired using the
same enzyme systems that are used in Base-Excision Repair (BER). - BER, NER and MMR are all strand specific SSB repairing system
REPAIRING STRAND BREAKS
Double-Strand Breaks (DSBs)
There are two mechanisms by which the cell attempts to repair a
complete break in a DNA molecule:
- Direct joining of the broken ends. This requires proteins that recognize and bind
to the exposed ends and bring them together for ligating. They would prefer to
see some complementary nucleotides but can proceed without them so this
type of joining is also called Nonhomologous End-Joining (NHEJ). - Homologous Recombination (next page)
Direct joining
Direct joining of the broken ends. This requires proteins that recognize and bind
to the exposed ends and bring them together for ligating. They would prefer to
see some complementary nucleotides but can proceed without them so this
type of joining is also called Nonhomologous End-Joining (NHEJ)
REPAIRING OF DOUBLE STRAND BREAKS
Homologous Recombination (also known as Homology-
Directed Repair HDR).
Sister chromatids
available in G2 after chromosome duplication
REPAIRING OF DOUBLE STRAND BREAKS
Homologous Recombination (also known as Homology-
Directed Repair HDR).
Homologous chromosome
(in G1; that is, before each chromosome
has been duplicated). This requires searching around in the nucleus
for the homolog - a task sufficiently uncertain that G1 cells usually
prefer to mend their DSBs by NHEJ.
REPAIRING OF DOUBLE STRAND BREAKS
Homologous Recombination (also known as Homology-
Directed Repair HDR).
Same chromosome
if there are duplicate copies of the gene on the
chromosome oriented in opposite directions (head-to-head or back-to-
back).
REPAIRING OF DOUBLE STRAND BREAKS
Homologous Recombination (also known as Homology-
Directed Repair HDR).
Two of the proteins used in homologous recombination are encoded by
the genes _______ and _______. Inherited mutations in these genes
predispose women to ______ and _________ cancers
Two of the proteins used in homologous recombination are encoded by
the genes BRCA1 and BRCA2. Inherited mutations in these genes
predispose women to breast and ovarian cancers
Gap O G0
quiescent/ Senescent
Gap 0 G0
A resting phase where the cell has left the cycle and has stopped
dividing
Interphase
Gap 1
Gap 1 G1
Cells increase in size in Gap 1. The G1 checkpoint control
mechanism ensures that everything is ready for DNA synthesis.
Interphase
Synthesis
Synthesis S DNA replication occurs during this phase.
Interphase
Gap 2 or G2
Gap 2 G2
During the gap between DNA synthesis and mitosis, the cell will
continue to grow. The G2 checkpoint control mechanism ensures that
everything is ready to enter the M (mitosis) phase and divide.
Cell
division
M mitosis
-Cell
division
-Mitosis M
Cell growth stops at this stage and cellular energy is focused on the
orderly division into two daughter cells. A checkpoint in the middle
of mitosis (Metaphase Checkpoint) ensures that the cell is ready to
complete cell division.
MAJOR PROTEINS THAT CONTROL CELL CYCLE
Control Proteins
- Cyclin-dependent protein kinases (Cdks)
- Cyclins
MAJOR PROTEINS THAT CONTROL CELL CYCLE
Complexes: Cdk-cyclin
- ability of Cdk to “P” target is dependent on the cyclin that it forms a
complex with
CELL CYCLE CHECKPOINT PROTEINS
G1 checkpoint
- Active cyclin D-cdk4 complexes phosphorylate retinoblastoma protein (pRb) in the
nucleus. Unphosphorylated Rb acts as an inhibitor of G1 by preventing E2F-mediated
transcription. - Activated P53-p21 interaction also prevent G1 to S phase transition
CELL CYCLE CHECKPOINT PROTEINS
G2 checkpoint
- DNA damage in the G2 phase initiate a signaling cascade that regulates wee1 and
cdc25 activity, therefore controlling mitotic entry via cyclin B-cdK2 can delay in
mitotic entry
CELL CYCLE CHECKPOINT PROTEINS
Mitotic checkpoint
- Spindle assembly checkpoint (SAC), through P53-cdK2-GADD45 cascade, to ensure
chromosome segregation is correct. - prevents anaphase onset until all chromosomes are properly attached to the spindle.
APOPTOSIS
The process of programmed cell death that may occur in _______________
The process of programmed cell death that may occur in multicellular
organisms
APOPTOSIS
Biochemical events lead to characteristic cell changes (morphology) and death
(different from necrotic or autophagic cell death):
- blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal
DNA fragmentation, and global mRNA decay
APOPTOSIS
- Highly _________ and ____________ process that confers advantages during an
organism’s lifecycle.
- Highly regulated and controlled process that confers advantages during an
organism’s lifecycle.
APOPTOSIS
Apoptotic Genes
- AIFM2, BAK1, BBC3, BCL2, BCL2L1 (BCL-X), BID, BNIP3, CDKN2A (p16INK4), DNM1L, MPV17, PMAIP1
(NOXA), SFN (14-3-3s), SH3GLB1, SOD2, TP53.
NECROSIS VS APOPTOSIS
Necrosis
- Increase in cell volume
- Loss of plasma membrane integrity
- Leakage of cellular contents
cells die accidentally due to injury,
trauma (ex. a poisonous spider bite),
or lack of nutrients (ex. lack of blood
supply)
NECROSIS VS APOPTOSIS
Apoptosis
- cell shrinkage
- plasma membrane blebbing
- formation of apoptotic bodies
Cells commit suicide when lacking any
incoming survival, or severe viral infection.
or when they detect extensive DNA
damage in their own nucleus. Cells will
murder other cells to clear out unneeded
cells or to eliminate potentially self-attacking
immune cells.
THE UBIQUITIN-PROTEASOME SYSTEM
Proteasome, a complies inside all
Proteosome, a complexes inside all eukaryotes and archaea, and in some
bacteria.
THE UBIQUITIN-PROTEASOME SYSTEM
Proteosome main function
Main function is to degrade unneeded or damaged proteins by proteolysis, a
chemical reaction that breaks peptide bonds
THE UBIQUITIN-PROTEASOME SYSTEM
Proteasomes are located both in the
nucleus and in the cytoplasm
THE UBIQUITIN-PROTEASOME SYSTEM
The proteasomal degradation pathway is essential for many
Cellular processes,
including the cell cycle, the regulation of gene expression, and responses to
oxidative stress.
THE UBIQUITIN-PROTEASOME SYSTEM
Protein degradation during
anaphase of mitosis of cell cycle.
THE UBIQUITIN-PROTEASOME SYSTEM
The importance of proteolytic degradation inside cells and the role of ubiquitin
in proteolytic pathways (UPP) was acknowledged in the award of the 2004
Nobel Prize in Chemistry to Aaron Ciechanover, Avram Hershko and Irwin Rose
HUMAN GENOME PROJECT
Launched in
1989 -expected to take 15 years
HUMAN GENOME PROJECT
Competing Celera project launched in
1998
HUMAN GENOME PROJECT
Genome estimated to be ______ complete
* 1st Draft released in _____
* “Complete” genome released in _____
* Sequence of last chromosome published in _____
Genome estimated to be 92% complete
* 1st Draft released in 2000
* “Complete” genome released in 2003
* Sequence of last chromosome published in 2006
HUMAN GENOME PROJECT
cost:
Celera:
- Cost: rv $3 billion
- Celera: rv $300 million
THE HUMAN GENOME:
All humans share _____ of the same genetic sequence
99.9%
THE HUMAN GENOME:
____ of human genome variation comes from Single Nucleotide
Polymorphisms (SNPs)
90%
THE HUMAN GENOME:
The most common sources of variation between humans are
The most common sources of variation between humans are single
nucleotide polymorphisms (SNPs)—single base differences between
genomic sequences.
GENOME SEQUENCING PROJECT FINDS SNPS
The Human Genome Project involves sequencing
The Celera sequence comes from
DNA cloned from a number of
different people.[The Celera sequence comes from 5 people]
GENOME SEQUENCING PROJECT FINDS SNPS
_____ occur normally throughout a person’s DNA.
SNPs
GENOME SEQUENCING PROJECT FINDS SNPS
It occur almost once in every _______ nucleotides on average, which means
there are roughly 2-10 million SNPs in a person’s genome.
1,000
GENOME SEQUENCING PROJECT FINDS SNPS
This inevitably leads to the discovery of any sequence difference-_____ is the most
common one.
SNPs
GENOME SEQUENCING PROJECT FINDS SNPS
SNP can act as biological markers, helping scientists locate
that are
associated with disease
GENOME SEQUENCING PROJECT FINDS SNPS
When SNPs occur within a gene or in a regulatory region near a gene, they
may play a more direct role in ______________
disease by affecting the gene’s function
POLYMORPHISMS
Most disease-causing gene mutations are uncommon in the
______________.
Most disease-causing gene mutations are uncommon in the
general population.
POLYMORPHISMS
Polymorphisms definition
Genetic alterations that occur in more than 1 percent of the
population are called polymorphisms.
POLYMORPHISMS
They are common enough to be considered a normal variation in the
DNA
POLYMORPHISMS
Polymorphisms are responsible for many
of the normal differences
between people such as eye color, hair color, and blood type.
POLYMORPHISMS
Although many polymorphisms have no negative effects on a person’s
health, some of these variations may influence
he risk of developing
certain disorders.
Human Genetic Variation
what disease are caused by dysfunctional alleles
genetic diseases such as cystic fibrosis or Huntington’s disease are caused
by dysfunctional alleles
Human Genetic Variation
But there are different forms of each gene - known as
Alleles
blue vs. brown eyes (or purple vs white flowers
LIFE CYCLE OF SNP
(LONG WAY FROM MUTATION TO SNP)
1.) Appearance of
new variant
by mutation
2.) Survival of rare allele
3.) Increase in allele frequency
after population expand
4.) New allele is fixed
in population as novel polymorphism
SEQUENCE VARIATION AND SNP DISTRIBUTION
types of regions
- Non-Coding region (2/3)
- Regulatory region
- Coding region (1/3)
SEQUENCE VARIATION AND SNP DISTRIBUTION
coding region can be what or what
- Synonymous
- Non-Synonymous
SEQUENCE VARIATION AND SNP DISTRIBUTION
- Non-Synonymous
- missense
- Conservative
- Non-Conservative
- nonsense
SEQUENCE VARIATION AND SNP DISTRIBUTION
- One half of all coding sequence SNPs result in
non-synonymous
codon changes.
LEARNING FROM OUR DIFFERENCES
- Most common diseases and many drug responses have been
shown to be influenced by inherited differences in our genes - Studying genetic variances can improve our understanding
and treatment of disease
3 billion genes but only how many coding region genes
20,000
Polymorphisms dont really cause any changes to your
Body
Endogenous
In the body mutations
Cellular metabolism
Hydrolysis
Nuclease Digestion
Types of DNA damage
Single strand DNA damage
Double stranded DNA damage
Pyrimidine dimer such as Thymidine can cause sun mutations
DNA glycosylase
Generated at the AP site and add the correct base back at the 3’prime end and then DNA ligase can correct short strand
Thymidine dimer is what type of correction
NER
XP proteins cut from the _______ end and then polymerase ___________ and ___________ fix it
5’ end
Delta and Epsilon fix it
Mismatch repair
If polymerase didn’t find wrong one
Muts and mutsH are associated with
Lower organisms systems
If enzymes are not corrected then certain diseases can occur such as
Xeroderma pigmentoserum
Fanconi anemia
Blood syndrome
Ataxia telangietasia
BRCA1 is a
Homologenous double stranded repairing enzyme
BRCA2 is a
Suppressor gene that can lead to brest cancer and ovarian cancers
The cell cycle check phases
G1, G2, metaphase
Cyclin is activated by
Phosphorylation
P53 is a
Tumor suppressor gene
BCL2 is a
Mitochondrial protein and has regulatory functions
When the nucleus becomes fragmented what proteins are produced
uPP proteins and bind to and conjugate then go to proteasome so that degradation can occur
Inactivation of CDK by
Ubq is conjugated to it then cyclin- CDK will be broken and cyclin will go through apoptosis
90% of all human variation comes from
Single nucleotide polymorphisms
almost every 1000 bases you will find one different ________
SNP( variant) or different forms of each gene alleles such as hair and eye color
You can have two alleles but maybe only one ________
gene different per train on a chromonsome
Human Genetic Variation
Every human has essentially the same set of
Genes
