Lecture 4: Errors, Repair, SNP Flashcards

1
Q

Types of DNA damage

A
  • Single strand breaks
  • Double strand breaks
  • Pyrimidine Dimer
  • Nucleotide base oxidation
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2
Q

Cause of DNA damage

Exogenous

A

Thermal disruption
UV light exposure
Ionizing radiation
Exposure to mutagens, carcinogens, and viruses

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3
Q

Cause of DNA damage

Endogenous

A

Cellular metabolism
Hydrolysis
Nuclease Digestion

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4
Q

AGENTS THAT DAMAGE DNA

Certain wavelengths of radiation

A
  • 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.
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5
Q

AGENTS THAT DAMAGE DNA

Chemicals in the environment

A
  • 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
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6
Q

AGENTS THAT DAMAGE DNA

Intrinsic Spontaneous mutation

A
  • 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.
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7
Q

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

A

130

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8
Q

REPAIRING DAMAGED BASES

  • Damaged or inappropriate bases can be repaired by several mechanisms:
A
  • 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)
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9
Q

REPAIRING DAMAGED BASES

Excision Repair

A

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.

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10
Q

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).

A
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11
Q

DIRECT REPAIR GENES

DNA photolyase

A
  • Natural repair system for pyrimidine dimers caused by UV damage
  • Directly reverse cyclobutane pyrimidine dimer (CPD) via photochemical reactions
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12
Q

DIRECT REPAIR GENES

O6-methylguanine-DNA methyltransferase (MGMT)

A
  • 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
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13
Q

BASE EXCISION REPAIR (BER)

The damaged base estimated to occur some _______ times a day in each cell in
our body!

A

20,000

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14
Q

BASE EXCISION REPAIR (BER)

Remove it by a __________. There are at least 8 genes encoding different
DNA glycosylases:

A

Remove it by a DNA glycosylase. There are at least 8 genes encoding different
DNA glycosylases

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15
Q

BASE EXCISION REPAIR (BER)

There are at least 8 genes encoding different
DNA glycosylases.

A
  • 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
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16
Q

BASE EXCISION REPAIR (BER)

Replacement with the correct nucleotide. This relies on DNA polymerase _________, one of at least _____ DNA polymerases encoded by our genes

A

Replacement with the correct nucleotide. This relies on DNA polymerase
beta, one of at least 11 DNA polymerases encoded by our genes

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17
Q

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.

A

Ligation of the break in the strand. Two enzymes are known that can do this;
both require ATP to provide the needed energy.

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18
Q

BASE EXCISION REPAIR

One stand of DNA contains __________
base, such as _________

A

One stand of DNA contains deaminated
base, such as Uracil

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19
Q

BASE EXCISION REPAIR

DNA glycosylases scans _______

A

the DNA and
removes Uracil, leaving AP site

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20
Q

BASE EXCISION REPAIR

AP endonuclease locates _________

A

AP site and nicks backbone

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21
Q

BASE EXCISION REPAIR

DNA Exonuclease removes

A

nucleotides near the nick, leaving gap

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22
Q

BASE EXCISION REPAIR

DNA Polymerase synthesizes to _______

A

fill in gap

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23
Q
A
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24
Q

BASE EXCISION REPAIR

DNA Ligase seals the

A

backbone

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25
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.
26
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)
27
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'
28
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
29
XERODERMA PIGMENTOSUM (XP) Type of disease
A rare inherited autosomal recessive disease of humans in which a deficiency of excinuclease occurs
30
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
31
XP resulting in
* XP resulting in skin discolouration and multiple tumours on exposure to UV light.
32
XP unprepared __________ in humans may lead to ___________
* Unrepaired pyrimidine dimers in humans may lead to melanoma
33
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
34
MISMATCH REPAIR (MMR) Mismatch repair deals with correcting mismatches of the __________; that is, failures to maintain normal ________ base pairing (A*T, C*G)
Mismatch repair deals with correcting mismatches of the normal bases; that is, failures to maintain normal Watson-Crick base pairing (A*T, C*G)
35
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.
36
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
37
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.
38
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
39
Mismatch Repair in Prokaryotes and Eukaryotes Mut S recognizes
and binds mismatch
40
Mismatch Repair in Prokaryotes and Eukaryotes Mut L links
S to H
41
Mismatch Repair in Prokaryotes and Eukaryotes Mut H recognizes the
CH3-parental strand and makes nick on daughter strand
42
Mismatch Repair in Prokaryotes and Eukaryotes In human: MutS =
hMSH (1-6); MutL = hMLH1 and hPMS2; MutH = GTBP
43
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
44
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)
45
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)
46
REPAIRING OF DOUBLE STRAND BREAKS Homologous Recombination (also known as Homology- Directed Repair HDR). Sister chromatids
available in G2 after chromosome duplication
47
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.
48
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).
49
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
50
Gap O G0
quiescent/ Senescent Gap 0 G0 A resting phase where the cell has left the cycle and has stopped dividing
51
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.
52
Interphase Synthesis
Synthesis S DNA replication occurs during this phase.
53
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.
54
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.
55
MAJOR PROTEINS THAT CONTROL CELL CYCLE Control Proteins
* Cyclin-dependent protein kinases (Cdks) * Cyclins
56
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
57
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
58
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
59
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.
60
APOPTOSIS The process of programmed cell death that may occur in _______________
The process of programmed cell death that may occur in multicellular organisms
61
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
62
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.
63
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.
64
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)
65
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.
66
THE UBIQUITIN-PROTEASOME SYSTEM Proteasome, a complies inside all
Proteosome, a complexes inside all eukaryotes and archaea, and in some bacteria.
67
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
68
THE UBIQUITIN-PROTEASOME SYSTEM Proteasomes are located both in the
nucleus and in the cytoplasm
69
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.
70
THE UBIQUITIN-PROTEASOME SYSTEM Protein degradation during
anaphase of mitosis of cell cycle.
71
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
72
HUMAN GENOME PROJECT Launched in
1989 -expected to take 15 years
73
HUMAN GENOME PROJECT Competing Celera project launched in
1998
74
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
75
HUMAN GENOME PROJECT cost: Celera:
* Cost: rv $3 billion * Celera: rv $300 million
76
THE HUMAN GENOME: All humans share _____ of the same genetic sequence
99.9%
77
THE HUMAN GENOME: ____ of human genome variation comes from Single Nucleotide Polymorphisms (SNPs)
90%
78
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.
79
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]
80
GENOME SEQUENCING PROJECT FINDS SNPS _____ occur normally throughout a person’s DNA.
SNPs
81
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
82
GENOME SEQUENCING PROJECT FINDS SNPS This inevitably leads to the discovery of any sequence difference-_____ is the most common one.
SNPs
83
GENOME SEQUENCING PROJECT FINDS SNPS SNP can act as biological markers, helping scientists locate
that are associated with disease
84
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
85
POLYMORPHISMS Most disease-causing gene mutations are uncommon in the ______________.
Most disease-causing gene mutations are uncommon in the general population.
86
POLYMORPHISMS Polymorphisms definition
Genetic alterations that occur in more than 1 percent of the population are called polymorphisms.
87
POLYMORPHISMS They are common enough to be considered a normal variation in the
DNA
88
POLYMORPHISMS Polymorphisms are responsible for many
of the normal differences between people such as eye color, hair color, and blood type.
89
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.
90
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
91
Human Genetic Variation But there are different forms of each gene - known as
Alleles blue vs. brown eyes (or purple vs white flowers
92
93
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
94
SEQUENCE VARIATION AND SNP DISTRIBUTION types of regions
* Non-Coding region (2/3) * Regulatory region * Coding region (1/3)
95
SEQUENCE VARIATION AND SNP DISTRIBUTION coding region can be what or what
* Synonymous * Non-Synonymous
96
SEQUENCE VARIATION AND SNP DISTRIBUTION * Non-Synonymous
* missense - Conservative * Non-Conservative - nonsense
97
SEQUENCE VARIATION AND SNP DISTRIBUTION * One half of all coding sequence SNPs result in
non-synonymous codon changes.
98
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
99
3 billion genes but only how many coding region genes
20,000
100
Polymorphisms dont really cause any changes to your
Body
101
Endogenous
In the body mutations Cellular metabolism Hydrolysis Nuclease Digestion
102
Types of DNA damage
Single strand DNA damage Double stranded DNA damage Pyrimidine dimer such as Thymidine can cause sun mutations
103
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
104
Thymidine dimer is what type of correction
NER
105
XP proteins cut from the _______ end and then polymerase ___________ and ___________ fix it
5' end Delta and Epsilon fix it
106
Mismatch repair
If polymerase didn't find wrong one
107
Muts and mutsH are associated with
Lower organisms systems
108
If enzymes are not corrected then certain diseases can occur such as
Xeroderma pigmentoserum Fanconi anemia Blood syndrome Ataxia telangietasia
109
BRCA1 is a
Homologenous double stranded repairing enzyme
110
BRCA2 is a
Suppressor gene that can lead to brest cancer and ovarian cancers
111
The cell cycle check phases
G1, G2, metaphase
112
Cyclin is activated by
Phosphorylation
113
P53 is a
Tumor suppressor gene
114
BCL2 is a
Mitochondrial protein and has regulatory functions
115
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
116
Inactivation of CDK by
Ubq is conjugated to it then cyclin- CDK will be broken and cyclin will go through apoptosis
117
90% of all human variation comes from
Single nucleotide polymorphisms
118
almost every 1000 bases you will find one different ________
SNP( variant) or different forms of each gene alleles such as hair and eye color
119
You can have two alleles but maybe only one ________
gene different per train on a chromonsome
120
Human Genetic Variation Every human has essentially the same set of
Genes