Lecture 1: Functions and Dysfunctions Flashcards
What are the roles of the nucleus?
- Cell regulation
- Cell proliferation
- DNA transcription
What percentage is then nucleus of the cell?
6%
Central Dogma
DNA–> RNA–> Proteins
DNA replication occurs in a process called
Mitosis
What is meiosis?
Meiosis is the transfer of genetic information from parent–> child
What is the exclusive carrier of information from DNA–> protein?
mRNA
DNA is _____ stranded
double
What does it mean that DNA is anti-parallel?
One strand is 5’–>3’, the other stand is 3’–>5’
Base pairs are connected using ______ bonds.
Hydrogen
Phosphate is connected to sugar using ______ bonds
phosphodiester
Purine bases
A & G
Pyrimidine bases
C & G
A _____ bonds to ___
A double bonds to T
C _____ bond to ____
C triple bonds to G
Backbone of DNA
negatively charged sugar-phosphate backbone
An incredible amount of condensation is needed throughout the cell cycle, especially during _____. Why?
Mitosis. Because you’re passing on genetic information to offspring.
Mitotic chromosomes are condensed ____ times when compared with ______ chromosomes. Why?
Mitotic chromosomes are condensed 500 times when compared with interphase chromosomes to prevent damage to DNA as the chromosomes are separated and passed on to daughter cells.
Characteristics of Histone proteins?
- 20% of histone proteins are either lysine or arginine (many +++ charges)
- Attracted to - - - charged DNA backbone
- Lysine in histone proteins are target of post-translation modifications (PMT)
_____ in histone proteins are targets of post translational modifications (PMT)
Lysine
________ are highly conserved across species
histone proteins
What are the basic unit of chromosome packing?
Nucleosomes
Nucleosomes
Nucleosomes are the basic unit of chromosome packing. They are a sequence of DNA wrapped around an octomer of histone proteins.
Chromatin
Protein and DNA.
Forms beads on a string.
What are the two types of proteins that bind to DNA?
histone proteins and non histone chromosomal proteins
What is euchromatin?
Euchromatin is a LIGHTLY packed form of chromatin (DNA, RNA and protein), thus it is often under active transcription.
It is highly enriched in genes .
What is the most active portion of the genome?
euchromatin
92% of the human genome is ________
euchromatic. the remainder is called heterochromatin.
Heterochromatin
Very condensed chromatin that is highly concentrated at the centromeres and telomeres of chromosome. Packed very tight so not active at all: it is thought to be late replicating and genetically inactive. 8% of human genome
How does heterochromatin stain throughout the cell cycle?
Heterochromatin stains darkly throughout the cell cycle, even when in interphase.
Can heterochromatin be affected by gene expression?
No. There are very few active genes and those that are present are resistant to gene expression.
What is the position effect?
The position effect says that the activity of a gene is determined by its position on the chromosome.
Based on the position effect, what happens if a active gene is moved near heterochromatin?
It is silenced
What is found on chromosomes?
Genes and interspersed DNA that does not contain genes. It is regulatory information that used to be called “junk DNA”, however now it has vital roles.
The word gene was first used by _________, based on a concept developed by ___________
Wilhelm Johannsen, based on a concept developed by Gregor Mendel
______ only account for 1.5 of the whole genome.
Exons. Thus, only 1.5% of the genome is responsible for coding.
Before we make mRNA, our pre-mRNA must be spliced!
People usually have ______ differences in their genome, called __________.
1000
copy number variations (CNVs).
They are the reason for our differences and disease states.
How can we detect differences and abnormalities in genomes ?
Comparative Genome Hybridization (CGHs)
How does comparative genome hybridization work (CGH)?
CGH’s detect copy number variations (CNVs)
We probe a human genome CHIP with DNA from one person with the DNA from a “normal person” as reference..
What is RNAi?
RNAi is a process where RNA molecules (miRNA) inhibits gene expression or translation, neutralizing targeted mRNA molecules
RNA (miRNA) are used to regulate the activity of mRNA molecules
What are long terminal repeats (LTRs)?
LRTs are identical sequences of DNA that are repeated hundreds or thousands of times. Viruses use them to insert their genetic material into a host genome.
Where are LTRs found?
LTRs are found at either end of retrotransposons (proviral DNA).
How are LTRs formed?
They are formed by the reverse transcription of retroviral RNA.
What is the RNAi process of how they’re formed ?
- miRNA precursor comes back and folds back on itsellf.
- An enzyme called Dicer will come and degrade the dsRNA, cutting it into shorter fragments.
- One strand of the dsRNA is degraded, the other strand (called miRNA) associates with proteins.
- The bound miRNA binds to a target with the complimentary sequence.
- miRNA complex will then prevent gene expression by or blocking its translation.
Alternative RNA splicing occurs when? ______ come together and ______ Are spliced out
Alternative RNA splicing occurs prior to mRNA is formed
Exons come together.
Introns are spliced out.
:)
:)
99% of introns begin with a ____ and end with a ____.
Introns begin with a […GT] and end with a [AG…]
What percent of mutations affect RNA splicing?
15%
Acetylation regulates the _____ residues of histones.
Lysine
What does histone deacetylase do (HDAT)?
They deacetylate lysine residues on histones, causing the chromatin to be compact and transcriptionally repressed (OFF)
What does histone acetyl transferase do (HAT)?
HATs acetylate lysine residues, opening up & unwinding chromatin and making it transcriptionally active ( ON)
Pick one: HDAT/HAT
The beads are wound loosely on the string?
HAT
Pick one: HDAT/HAT
The beads are wound tight on a string
HDAT
Histones can be ______ and __________.
acetylated and deacetylated
Histone proteins can undergo ___________
post-translational modifications, altering their interactions with DNA and nuclear proteins.
Histone protein PTM
Histone proteins can undergo post-translational modifications (PTM), which alter their interactions with DNA and nuclear proteins. PTM mainly occurs on the tails of H3 and H4, which protrude from the nucleosome. The core of H2A and H2B can also be modified. Most targets of PTMs are [lysine and arginine residues] and phosphorylation of serine and threonine residues.
What are the main targets of PTM on the histone tails?
PTM usually target [lysine and arginine] resides on the histone tail and phosphorylation of serine and threonine residues.
Can the core of the histones H2A and H2B also be modified?
Yes.
What other types of post-translational modifications can histones undergo?
- Methylation
- Acetylation
- Phosphorylation
- Ubiquitination
- SUMOlation
- Citrullination
- ADP-ribolyation
which target Lys and Arginine residues.
What is DNA Methylation?
DNA methylation is when methyl groups are added to C[ytosine] and A[denine] residues of DNA molecules. It changes the activity of the DNA segment without changing the sequence. It is necessary for development.
What happens when DNA methylation occurs at a gene promoter?
It supresses gene transcription.
METHYLATION MAKES DNA MUTE
(mutes transcription)
What residues does DNA methylation occur on?
Adenine and cytosine
DNA methylation and its relevance to cancer
Hypermethylation at CpG (promoter) causes transcriptional silencing (inherited by daughter cells) can also silence tumor suppressors which will not inhibit cancer growth.
DNA hypomethylation
DNA hypomethylation causes
- chromosomes to be unstable
- loss of imprinting.
DNA hypermethylation can cause what?
DNA hypermethylation can cause silencing of gene promoters
In order for DNA Replication to occur, the 2 parental strands must be _______
separated.
What does it mean that DNA replication is semi-conservative?
Each strand in a double helix acts as a template for the synthesis of a new, complimentary strand.
DNA dependent-DNA polymerase makes the new strand in the ________ direction
5’–>3’
DNA polymerase requires what?
A template and a primer with a free 3’-OH. (Primase creates and RNA primer)
___,____,____,____ are all needed
dATP, dGTP, dCTP and dTTP
DNA Replication produces what?
2 identical DNA strands, each with one old and one new strand.
Can DNA polymerase make DNA from scratch?
No, they need a primer.
Besides synthesizing DNA, what else can DNA polymerase do?
DNA polymerase has the ability to proofread itself.
Where does DNA replication begin & how?
DNA replication begins at the origin of replication, which are recognized by their sequence. DNA is opened and replication forks are formed, forming the replication bubble. As replication proceeds, the replication forks move in opposite directions.
DNA replication is semi-discontinuous. What does this mean?
DNA polymerase can only synthesize in the 5’–>3’ direction. thus, the leading strand is synthesized continuously and the lagging strand is made discontinuously in okazaki fragments.
DNA helicase
DNA helicase is a protein with 6 identical subunits.
Using ATP, it will induce a conformational change, propel like an engine and UNWIND the helix at 1000 bp/sec
How fast does helicase move?
1000 bp/sec
ssBinding Proteins
ssBP will bind to the exposed ssDNA and help stabilize the unwound DNA from forming hairpin loops.
What does topoisomerase do?
A REVERSIBLE enzyme that
- breaks phosphodiester bonds
- relieves supercoiling.
What are topoisomerases called in bacteria?
DNA gyrase
Which DNA replication player is targeted by anti-cancer drugs?
Topoisomerase.
Why do topoisomerase inhibitors act as anti-cancer agents? (4)
They prevent cancer cells from replicating by:
- They block the cell cycle because they are not able to keep the DNA unwound.
- They generate single/double stranded breaks
- Harms integrity of genome
- Leads to apoptosis and cancer cell death
Why do germ cells and somatic cells have low mutation rates? 1 mistake in every 10^9 base pairs is seen
DNA polymerase can proofread. If they are not caught, errors are further corrected by post-replication repair mechanisms
Topoisomerase I inhibitor
Irnotecan- used to tx colorectal cancer
Topoisomerase II inhibitor
Etoposisde
Anthracyclines like doxorubocin and daunoribocin
Etoposide causes what?
secondary leukemias
Anthracyclines cause what?
cardiotoxicity
What DNA damage occurs by UV radiation?
Covalent linkage between 2 adjacent pyrimidines (T-T and C-T), called pyrimidine dimers
What DNA damage occurs by ionizing radiation?
Double stranded breaks
What DNA damage occurs by non-ionizing radiation?
Adjacent thymine dimers form
What are the two types of DNA damage that occur spontaneously?
- Depurination
2. Deamination
Depurination
Depurination is the loss of a A/G. 5000 are lost a day
Deamination
amino group of a purine or pyrimidine base is hydrolyzed
Most common: C –> U. Occurs in about 100 bases a day.
Deamination of adenine can make
hypoxanthine
Deanimation of guanine can make
xanthine
Deamination of cytosine can make
uracil
What are the possible outcomes of spontaneous DNA damage?
- DNA Replication can follow
2. Base can be deleted or substituted
What is a CpG site?
regions of adjacent cytosine-[phosphate]-guanine groups
:
:
Are methylated cytosine residues in CpG sites/islands bad?
Yes. Methylation will stably silence the gene. This can produce cancer if it happens in a DNA repair gene.
Is DNA repair effective for CpG islands/sites?
DNA repair is relatively ineffective. Only 3% of C nucleotides are methylated but it accounts for 1/3 of all point mutations.
What is the risk of eating well done meat compared to not well done?
Well done meat produces a carcinogen, BPDE (a epoxide), which was originally a pro-carcinogen, benzo[a]pyrene.
What are cross-linking agents that can cause DNA damage?
- Nitrogen Mustard (a war drug)
Anti-cancer agents
2. Cisplatin
3. Mitomycin C
4 Carmustine
What are alkylating agents that can cause DNA damage?
- dimethyl sulfate (DMS)
2. methyl methanesulfonate (MMS)
What is an important intercalating agent (in between DNA bases) that can damage DNA?
Thalidomide- Rx for morning sickness but caused birth defects
DNA damage has variable outcomes in our bodies. What are these outcomes?
- They can be repaired & normal cell function
- Not repaired but still have no deleterious consequences
- Deleterious consequences can result in impaired cellular fx, cell death or mutations/genetic instability which can cause cancer.
What type of DNA damage is repaired by direct repair, and how?
Direct repair enzymatically reverses or repairs some types of DNA damage.
For example, pyrimidine dimers can be separated with the aid of visible light via DNA photolyase. It binds to the dimers and absorbs light to catalyze the breaking of bonds between the pyrimidine in a process called (photoreactivation).
What type of DNA damage is repaired by base excision repair and how?
Repairs [single base mismatches] and [small non-distorting alterations.]
- [DNA glycolases] detects altered base
- DNA glycolase will then remove the base by hydrolyzing the n-glycosidic bond.
- [AP endonuclease] cuts the phosphodiester bond.
- [AP lyase] removes the sugar-phosphate backbone.
- [DNA polymerase] replaces the excised nucleotides
- [DNA ligase] seals nick
What type of DNA damage is repaired by nucleotide excision repair (NER) and what are the enzymes/mechanism?
NER repairs [chemical adducts that distort DNA], such as -
- pyrimidine dimers,
- BPDE-guanine adducts
- cisplatin adducts.
- NER complex recognizes distortions and cuts DNA on both sides of damage.
- Its removed
- DNA polymerase fills i gap
- DNA ligase seals nick
Disorder involved with [NER]
Xeroderma pigmentosum
Mismatch excision repair (MER)
Types of damage repaired:
Enzyme/mechanism involved:
MER system repairs mismatched base.
- [MER complex] binds at mismatch
- [Endonuclease] cuts daughter strand
- [helicase] removes segment of DNA that has the mismatch from the daughter strand
- [Poymerase-sigma] fills in gap
- [DNA ligase] seals
Assx disorder with MER
Hereditary Nonpolyposis Colorectal Cancers
What are the two types of Recombination Repair mechanisms, and what is their general job?
Recombination repair occurs when double strand breaks, interstrand cross-linking. There are 2 mechanisms to fix:
- Non-Homolologous End Joining (NHEJ)-
- Homologous Recombination
Non-Homolologous End Joining (NHEJ)
Damaged segment is not replaced. Instead, the ends are joined together by DNA ligase., which repairs the double strand but several base pairs at the site of lesion are mission. This is the preferred mechnism.
Homologous Recombination
Repairs ds breaks that occur after DNA has replicated but before cell division so that replicated DNA helices are together. Genetic information from the undamaged duplex is used to repair the damaged duplex.
Disease assx with Recombination Repair
BRCA1/breastcancer
Cockayne syndrome is associated with what repair mechanism?
Trancription-coupled repair (TCR)
Bypass synthesis is also called
translesion synthesis
MER involves 2 proteins: _____ and _____ and they repair DNA
MutS and MutL
What do MutS and MutL do?
MutS binds to the chain
MutL will then scan for the nick and trigger degradation of the nicked strand
What is important to know about PTM’s?
PTM’s talk to one another. You can have things such as phosphoryl-dependent sumolation, etc.
Epigenetics
Regulating gene activity without altering the sequence.
Epigenetics are affected by what 5 factors:
- development
- environmental chemicals
- drugs/pharmaceuticals
- aging
- diet
HDACs are important for what?
anti-cancer agents
2 important HDAC inhibitors
- vorinostat
2. entinostat
What do trasncription factors do?
They talk to eachother, do not work alone!
–
What are the relevance of CpG islands around a start codon?
- 1/10 are CpG.
- 70% of promoters contain CpG islands
- They let the DNA know that Start is happening soon!
What happens in when CpG islands are methylated?
Methylation of CpG islands are stably silenced.
Difference between CpG islands and CpG sites?
CpG islands tend to occur near the promotor or regulatory region of genes.
CpG sites occur at random sites throughout the egnome.
Methylation of CpG islands occur where?
5 position of the pyrimidine ring of cytosine resides, forming 5-methylcytosines.
Are there a lot of HDACS?
Yes. HDACs are a large family of proteins.
