Function/dysfunction- Gene Reg Flashcards
What is the nucleus’s role in the cell
Cell regulation, proliferation, and DNA transcription
What is the central dogma of genetics
DNA —(transcription)—>RNA —(translation) —> Protein
What is the exclusive carrier of information from DNA to Protein ?
mRNA
T/F
RNA can be reverse transcribed into DNA
True
*reverse transcriptase relates to RNA Virus (HIV)
How many bonds are between A-T and C-G in double stranded DNA
AT is double (hydrogen) bond
GC is triple (hydrogen) bond
Why are mitotic chromosomes condensed 500 times more than interphase chromosomes
To prevent physical damage to the DNA as chromosomes are separated and passed to daughter cells
What bonds are between DNA and the histone octamers in each nucleosome
Hydrogen bonds
- hydrophobic interactions
- salt linkages
T/F
DNA is wound around histone Proteins
True
20% of histone protein amino acid residues are lysine or arginine
What is a nucleosome
Basic unit of chromosome packing:
- has 8 histone proteins - histone octamer = protein which DNA is wound around
Protein + DNA =
Chromatin
Euchromatin vs Heterochromatin
Euchromatin
- lightly packed
- undergoes transcription
- most active portion of genome
Heterochromatin
- very condensed
- stains darkly
- genetically inactive
- high concentrated at centromeres and telomeres
What is the position effect?
Activity of a gene depends on relative position on chromosome
- is actively expressed gene is relocated near Heterochromatin it will be silenced
T/F
Copy number variations are detected by comparative genome hybridization and are the basis for our differences as humans
True
What is RNAi
RNA interference
- miRNA inhibit gene expression or translation
- this happens by a miRNA precursor folds on itself and a Dicer enzyme cuts dsRNA into short segments. one strand is degraded and the single strand left is miRNA. The miRNA associates with a protein complex that prevents gene expression by degrading or blocking target mRNA
What are Long terminal repeats?
- identical sequences of DNA repeated
- found at retrotransposons (proviral DNA) ends
- formed by reverse transcription of retroviral RNA
- used by viruses to insert their genetic material into the host genome
What base pairs are found at intron/exon border?
GT is at beginning
AG is at end
- intron is spliced out info
- exon is the info kept
What gives gene diversity
Alternative RNA splicing
*exons and introns
What does HDAC and HAT do?
Histone Deacetylase actively represses gene expression
- keeps chromatin condensed
- have corepressor proteins
Histone acetyl transferase promotes gene expression
- allows chromatin to open and be transcribed
- has coactivator proteins
*both bind to promoter region
T/F
histone protein tails are the target of numerous Post-translational modifications
True
PTM’s occur at the n-terminus
What is DNA methylation ?
PTM
- methyl group added to DNA (cytosine and adenine) by methyl transferase enzymes
- represses gene transcription at gene promoter
What happens when CpG islands are hypermethylated
Transcriptional silencing which can be passed down to daughter cells
- can cause cancer if it is in DNA repair gene
- causes 5-methyl cytosine to become thymine
What does hypomethylation cause
Chromosomal instability and loss of imprinting
Which direction is new DNA synthesized
5’—>3’
DNA polymerase requires a primer with a _______ to begin processing
With a free 3’ -OH group
What unwinds DNA to begin DNA replication
**uses ATP
DNA helicase
What binds to exposed DNA to help stabilize unwound DNA and prevents hairpin formation in DNA replication
single-stranded DNA-binding protein (SSBP)
What does DNA polymerase do ?
Replicate DNA in 5’—>3’ direction, makes RNA-DNA primer, fills in gaps
What does endonuclease do in DNA replication
Remove RNA primers
What relieves supercoiling during DNA replication by breaking phosphodiester bond? And is the target for many pharm drugs for anti-cancer agents?
DNA topoisomerase
Topoisomerase inhibitors
1- irinotecan used in colorectal cancer
2- etoposisde
They block the cell cycle, generate breaks, lead to apoptosis and cancel cell death
T/F
DNA polymerase can proofread
True
What does UV radiation do to cause DNA Damage
Produces a covalent linkage between adjacent pyrimidines (T-T or C-T)
**pyrimidine dimers
Spontaneous DNA damage
Depurination- 5000 purine lost a day by hydrolysis
**leaves free OH end on remaining sugar phosphate
Deamination- C to U change 100 times a day (any base pair change)
Benzo(a)pyrene turns to BPDE (an epoxide) means what **in well done-grilled meats
Pro carcinogen turns in a known carcinogen
Examples of cross linking agents (DNA Damage by chemicals)
Nitrogen mustard
Cisplatin
Mitomycin C
Carmustine
Examples of alkylating agents ( DNA Damage by chemicals)
Dimethyl sulfate (DMS) Methyl methanesulfonate (MMS)
Example of intercalating agent (DNA damage by chemicals)
Thalidomide
What repair mechanism fixes pyrimidine dimers and methylguanine
Direct Repair (enzymatic repair)
What repair mechanism fixes single base mismatches
Base excision repair (BER)
What repair mechanism is associated with xeroderma pigmentosum
Nucleotide excision repair (NER)
What repair mechanism is associated with hereditary nonpolyposis colorectal cancer (HNPCC)
Mismatch excision repair (MER)
*fixes mismatched bases in daughter strand
What repair mechanism is associated with BRCA breast cancer
Recombination repair
What repair mechanism is associated with cockayne syndrome ?
Transcription coupled repair (TCR)
- RNA polymerase is permanently stalled at sites of damage in important genes
- causes growth retardation, skeletal abnormalities, and sunlight sensitivity
HDAC remove acetyl groups from the _____ on core histone and nonhiston proteins
Lysines
2 HDAC inhibitors
Vorinostat and entinostat
-anticonvulsives and anti cancer drugs
Embryonic stem cells vs adult stem cells
embryonic are pluripotent (capable of becoming any cell type)
Adult are undifferentiated cells that generate cells types in the tissues in which they reside (ex. Hematopoietic stem cells will produce blood cells only)
Stem cells differentiate into specialized cells in stages. These stages involve multiple facets that combine to produce epigenetic markers in the cells’s DNA that restrict DNA expression and thus the type of cell that the stem cell can become. Can this DNA expression be passed on to daughter cells?
Yes, through cell division
DNA Structure:
Where are the nucleotides linked in DNA?
The 3’ OH group is connected to 5’ phosphate group by a phosphodiester bond.
*every DNA strand has a phosphate residue at the 5’ terminus and a free hydroxyl group at the 3’ terminus
In no dividing stages of the cell cycle (G1, G0, G2, S) the transcriptionally active chromatin is called _____; the inactive chromatin is called ______; and in the M phase of the cell cycle chromatin becomes _____
- Euchromatin
- Heterochromatin
- Mitotic Chromosomes which are condensed 500 times more than interphase chromosomes to prevent physical damage to the DNA as they are separated and passed to daughter cells
T/F
The 2’ C of ribose in RNA has an OH group while DNA does not
True
Imprinting is an _____ process that involves the methylation and histone modification of egg or sperm cells during their formation while the genetic sequence is unchanged. Imprinting is duplicated in all somatic cells
Epigenetic
If Uniparental disomy occurs in imprinted genes (silenced genes) then essentially the child was passed on only 1 gene and it does not function therefore diseases occur such as
Prader-WIllie or Angelmans
What is the presence of more than one type of mitochondrial DNA (some normal some mutant) within a cell or organism. Symptoms do not occur until adulthood bc the cell must divide enough times before sufficient amount are present to cause problems
Heteroplasmy
What is topoisomerase in bacteria called
DNA gyrase
What does single-stranded DNA binding protein do in DNA replication?
Binds to the ss-DNA that has been separated to prevent them from reannealing
What does DNA ligase do in DNA replication
Seals nicks between Okazaki fragments
What does FLAP endonuclease 1 (FEN1) do in DNA replication ?
Removes RNA primers
What bonds does Helicase disrupt when it unwinds DNA at the replication fork ?
Hydrogen bonds between complementary nucleotides
What are telomeres and how are they shortened? What prevents shortening?
Telomeres cap DNA and are made of repeats of non-coding DNA. They are shortened after the removal of the last RNA primer from the 5’. Shortening limits the number of times cells can divide bc loss of telomeres triggers apoptosis.
Telomerase (reverse transcriptase) prevents shortening. Only in germ line and stem cells, not in somatic cells.
T/F
DNA synthesis involves the formation of 3’—>5’ phosphodiester bonds
True
_____- lack the 3’ -OH group and can act as a drug that inhibits DNA replication
Nucleoside analogues
Ex: Acyclovir Ara-C Cytarabine AZT
Nucleoside analogues need to be converted to _______ before they can inhibit DNA polymerase
dNTPS
Ara-C is a nucleoside analogue that contains the sugar arabinose and is converted by cells into ara-CTP. Hoe does Ara-CTP inhibits DNA synthesis ?
Ara-CTP is a potent competitive inhibitor of DNA polymerase
*used to treat leukemia
The nucleoside analogue AZT is used to treat what? How does it do this?
Used in HIV therapy.
-the drug is taken up by HIV-infected cells and is activated by viral reverse transcriptase. Because the AZT lacks a 3’OH group it stops viral DNA synthesis
What nucleoside analogue is used to treat herpes? How does it do this?
Acyclovir
- thymidine kinase catalyzes 1 phosphorylation
- the host’s enzyme converts the monophosphate into a triphosphate
- it lacks a 3’ OH group and therefore stops DNA synthesis during replication
Physical vs chemical agents that damage or mutate DNA
Physical agents
- ex: ionizing radiation and nonionizing radiation
- displace electrons from atoms
- sources of IR is x-rays and CT scans
Chemical agents
- react with functional groups on DNA
- intercalate between DNA bases
What is the term for a chemical agent that causes permanent damage to DNA
Mutagen
*most mutagens are carcinogens
Transition vs a transversion point mutation
Transition is a purine substituted as a purine and vice verse
Transversion is a purine substituted as a pyridimidine and vice versa
Two examples of spontaneous damage to DNA.
- Depurination
- purine base is removed via hydrolysis of N-glycosidic bond between base and deoxyribose
- results in a apurinic or abasic site in the DNA strand with free OH end - Deamination
- amino group of purine or pyrimidine base is hydrolyze such that adenine is converted to hypoxanthine and guanine is converted to xanthine and cytosine is converted to uracil.
Ionizing radiation is radiation-induced DNA damage. What changes are made to DNA as a result ?
- 40-60 chemically distinct base damages
- direct strand breaks
- DNA-protein cross-links
Nonionzing radiation (UV light) is a type of radiation -induced DNA damage. What changes are made to DNA as a result?
-damages DNA by promoting the formation of a covalent linkage between adjacent pyrimidine bases which results in 2 major photoproducts that are toxic, mutagenic, and carcinogenic
What are the two types of major photoproducts that are toxic, mutagenic, and carcinogenic created by non-ionizing radiation damage of DNA?
- Pyrimidine cyclobutane dimers (more common)
- 6-4 covalent linkage of 2 pyrimidines
- the mutations are present in the P53 gene from UV exposure and are associated with basal and squamous cell carcinomas
What are the two groups that broadly classify chemical agents that cause DNA damage ?
- Agents that require metabolic activation
- ex: benzo(a)pyrene, aflatoxin B1 - Agents that act directly to modify DNA
- ex: cross linking agents, alkylating agents, and intercalating agents
What does Benzo(a)pyrene do to damage DNA?
- chemical agent present in many combustion products (cig smoke, exhaust, charred meat)
- makes a bulky adduct with guanine and PREVENTS REPLICATION AND GENE EXPRESSION
- also inhibits P53
What does a cross-linking agent do to damage DNA?
- chemical agent
- forms cross-links between bases in same DNA strand or between complementary DNA
Ex: nitrogen mustard, cisplatin, carmustine
*well known chemo drugs
What do alkylating agents do to damage DNA?
- chemical agent
- induce methylated base changes and alkylation of phosphodiesters into phosphotriesters
Ex: DMS, MMS
What do intercalating agents do to damage DNA?
- chemical agent
- insert between stacked bases of the DNA double helix which causes some unwinding of the helix and separation of base pairs
Ex: ethidium bromide, anthracyline antibiotics
DNA repair mechanism: Direct repair (enzymatic repair) repairs what
- pyrimidine dimers
- O6 methylguanine
Uses DNA photolyase and methyguanine methyltransferase
DNA repair mechanism: base excision repair (BER) fixes what
Single base mismatches, and nondistoring alterations such as depurination
DNA repair mechanism: nucleotide excision repair (NER) fixes what
Chemical adducts that distort DNA (pyrimidine dimers, and adducts)
*related to xeroderma pigmentosum
DNA repair mechanism: mismatch excision repair (MER) fixes what
Mismatched base in daughter strand
*related to hereditary nonpolyposis colorectal cancer (HNPCC)
DNA repair mechanism: recombination repair (nonhomologous end joining and homologous recombination) fix what?
Double-strand breaks, interstrand cross-linking
*related to BRCA 1/2 breast cancer
BRCA 1 and BRCA 2 genes encode proteins that facilitate homologous recombination
DNA repair mechanism: transcription-coupled repair (TCR) fixes what?
Type of NER that is initiated when Stalled RNA polymerase during transcription occurs (not replication)
*related to cockayne syndrome
DNA repair mechanism: transversion synthesis (bypass synthesis) fixes what?
Unrepaired thymine dimers or apurini AP sites
Individuals with xeroderma pigmentosum have a defet in proteins of the ______ complex
NER complex (XP proteins)
- the NER complex recognizes distortions in DNA and marks them for removal
- DNA ligase fills in the removed gaps
Individuals with defective proteins in the ____ complex have increased susceptibility to hereditary nonpolyposis colorectal cancers
MER complex (MSH2 or MLH1)
- mutation of one allele causes increased susceptibility
- mutation in both promotes tumor development
- MER complex binds to deformed DNA from mismatched bases
- endonuclease cuts the strand, exonuclease removes strand, polymerase fills the gap, ligase seals the nick
Defects in the genes encoding ____ and ____ lead to cockayne syndrome.
ERCC-6 and ERCC-8 which are TCR proteins
How do xeroderma pigmentosum and cockayne syndrome differ in manifestation?
Patients with cockayne syndrome do not exhibit pigmentary skin changes. Xeroderma patients have changes in skin pigment
What disease is characterized by skin that is extremely sensitive to sunlight and prone to developing melanomas and squamous cell carcinomas due to ineffective XP proteins of the NER complex?
Xeroderma pigmentosum
-XP proteins help the NER complex fix cyclobutane thymine dimers in DNA caused by UV light
XP proteins help the NER complex fix _______ in DNA caused by UV light
Cyclobutane thymine dimers.
*defect leads to xeroderma pigmentosum
What rare autsomal recessive, congenital disorder that is characterized by developmental and neurological delay, photosesitivity, hearing loss, eye abnormalities, and progeria (premature aging) that is caused by mutant ERCC6 and ERCC8 genes that fail to repair DNA via TCR
Cockayne syndrome types A and B.
- A from ERCC6
- B from ERCC8- 70% of most cases
Mutations of the tumor suppressor genes BRCA 1 and BRCA 2 cause a 5-fold increase in developing ____ in women. Mutations in men also cause the same disease
Breast cancer
Mutations of BRCA 1 also associated with cervical, uterine, pancreatic, colon, testicular and prostate cancer
Mutations of BRCA 2 is also associated with increased risk of melanoma, pancreatic, stomach, gallbladder, and bile duct cancer in women
Post-translational covalent modification of: methylation
- transfer of one or more methyl groups from S-adenosine-L-methionine to lysine or arginine residues of histone proteins
- catalyzes by HMTs (histone methytransferases)
- mediates heterochromatin formation and participates in regulating gene expression of euchromatic sites
- REPRESSES GENE TRANCRIPTION
- CHANGES DNA ACTIVITY WITHOUT CHANGING SEQUENCE
Post-translational covalent modification of: SUMOylation
— major regulatory of protein function
- covalent attachment of a memeber of the SUMO (small ubiquitin like modifier) family to lysine residues in target proteins
- analogous to ubiquitination but involves addition of SUMOs
Post-translational covalent modification of: ubiquitination
- the addition of ubiquitin to a substrate protein
- can mark proteins for degradation, alter cellular location, affect activity, and promote/prevent protein interactions
*most often noted for the the proteasome pathway for protein destruction
Post-translational covalent modification of: histone acetylation/deacetylation
- enzymatic addition of acetyl group to histone lysines (COCH3) from acetyl Coenzyme A
- HATs are the enzyme that transfers acetyl group. (Histone acetyltransferases)
- HDACs are the opposite of HATs and inhibit HAT function
- acetylation = gene expression
- deactyletion = repressed gene expression
*involved in the regulation of transcription, gene silencing, cell cycle progression, apoptosis, DNA replication….
Post-translational covalent modification of: phosphorylation
-amino acid residues Serine and threonine are phosphorylated but protein kinases
What is Chargraffs rule
In DNA the number of Adenines = thymine and guanines = cytosines
Base pairing in DNA (between AT and CG) can be disrupted by what two things
Heat or alkaline pH
Noncovalent mechanisms that give DNA structure its stability
Hydrophobic interactions, van der walls forces, base stacking
DNA double helix is present mostly in what form
B form
*other forms are A and Z
What is a nucleosome
*SHOWS HOW DNA IS PACKED
It is DNA wrapped around pairs of four histones (8 TOTAL) by hydrogen bonds. (Hydrophobic interactions and salt linkages aid stability)
- nucleosomes are further packed into chromatin
- rememeber 20% of histone is lysine or arginine
____ is the protein around which DNA is wound
Histone Octamer
PTM (post-translational modification) of histones by methylation, acetylation, and phosphorylation, impact gene expression by altering ______
Chromatin structure
Fanconi anemia groups A-G is caused by what defect
Defect in DNA interstrand cross-link repair
*causes congenital abnormalities, leukemia, and genome instability
Ataxia telangiectasia is caused by what defect
Defect in the ATM protein
- ATM protein is a protein kinase activated by double-strand breaks
- there no breaks are fixed
*causes leukemia, lymphoma, gamma ray sensitivity, and genome instability
Thymine dimers are corrected by what
Direct repair
-photolyase fixes thymine dimer
In Mismatch Exicision repair: _____ binds while _____ scans for the nick and triggers degradation of the nicked strand
MutS binds
MutL scans
What is a mechanism for regulating gene activity independent of DNA sequence that determines which genes are turned on or off by using PTMs
Epigenetics
What factors affect epigenetics
Development Environment Drugs Aging Diet
