Functions/Dysfunctions Of Genomic Regulation Flashcards
After the cytosol, which organelle comprises the highest volume out of the hepatocytic organelles?
Mitochondria at 22%
Which cellular organelle comprises 6% of the volume of hepatocytic organelles and is responsible for cell regulation, proliferation, and DNA transcription?
Nucleus
What is the central dogma of genetics? What enzyme challenges this dogma?
DNA –> RNA –> protein
Reverse transcriptase (contained by RNA viruses like HIV) which can reverse transcribe RNA to DNA)
Describe DNA in terms of number of strands, parallel vs. anti-parallel, and what type of bonds are present
DNA is double stranded, anti-parallel, and contains hydrogen bonds
The purines are A and G, pyrimidines are T and C
Which bases bind together, and how many H bonds are present?
A – T (2 H bonds)
G — C (3 H bonds)
Which is more condensed - mitotic chromosomes or interphase chromosomes?
Mitotic chromosomes are condensed 500x more than interphase chromosomes
142 hydrogen bonds are formed between DNA and the _________ octamer in each nucleosome via ____________ interactions and ________ linkages
Histone
Hydrophobic
Salt
True or false: Histone proteins are highly variable across species
False - they are highly conserved!
Approximately 20% of histone protein amino acid residues are either _________ or _________, which are basic and positively charged
Lysine; Arginine
What is the significance of histones having a positive charge?
They are attracted to the negatively charged DNA backbone
____________ residues in histone proteins are the target of post translational modifications
Lysine
Genetic material is considered ___________ during interphase, which then condenses to form chromosomes during M phase
Chromatin
Each histone as an octamer made up of (H2A.H2B)2 (H3.H4)2, which are connected by a ______ linker
H1
____________ are the basic unit of chromosome packing
Nucleosomes
Proteins that bind to DNA are made up of what 2 major classes?
Histone proteins
Transcription factors (Non-histone chromosomal proteins)
Each nucleosome core particle consists of a complex of _______ histone proteins
Eight
A histone octamer is a protein around which DNA is wound. Protein + DNA = ______________, forming “beads on a string”
Chromatin
Which type of chromatin makes up 92% of the human genome and is the most active portion of the genome?
Euchromatin
Describe euchromatin
Light packed form of chromatin (DNA, RNA, and protein)
Highly enriched in genes
Often, but not always, under active transcription
Describe heterochromatin
Very condensed, stains darkly throughout the cell cycle
Late replicating and genetically inactive
Highly concentrated at centromeres and telomeres
Very few active genes, those that are present are resistant to gene expression
What is the position effect?
The activity of a gene depends on its relative position on the chromosome
Actively expressed genes will be _____________ if relocated near heterochromatin
Silenced
Interspersed in DNA that encodes RNA and proteins is what may be referred to as “junk DNA”, which gets transcribed into long non-coding RNA. What is contained in this non-coding region?
Regulatory information
The human genome consists of ______ chromosomes
46
What percentage of our genome is made up of protein coding sequences (exons)?
1.5%
Humans are very similar to many other organisms in our genome, what is it that makes us different?
Post translational modifications and alternative RNA splicing
People usually have 1,000 genetic differences called Copy Number Variations (CNVs), which are the basis for our differences and for disease states. How do we detect CNV’s?
Comparative Genome Hybridization (CGH)
[probe human genome chip with DNA from one person and with DNA from a normal reference DNA - detects copy number variation]
_________ is a biological process in which RNA molecules (miRNA) inhibit gene expression, or translation by neutralizing targeted mRNA molecules
RNAi
Long terminal repeats are _________ sequences of DNA that repeat hundreds or thousands of times. They are found at either end of ______________ (or proviral DNA) by reverse transcription. They are used by viruses to insert their genetic material into host genomes
Identical
Retrotransposons
microRNA:
The miRNA precursor folds back on itself, held together by _________ bonds.
An enzyme called ______ moves along the double stranded RNA, cutting it into shorter segments.
One strand of each short dsRNA is __________, the other strand (miRNA) associates with a complex of proteins.
The bound miRNA can base pair with any target mRNA that contains the _____________ sequence.
The miRNA protein complex prevents gene expression either by degrading the target mRNA or blocking its ___________.
Hydrogen Dicer Degraded Complementary Translation
With alternative RNA splicing, there are approximately _____ alternative splice products per gene. 99% of all introns begin with ________ and end with ________
Two
GT
AG
Numerous functional ________ appear in various proteins throughout the proteome, which drive protein-protein interactions
Motifs/domains
Histone Deacetylation utilizing histone deacetylase (HDAC) actively _____________ gene expression
Represses
[deacetylated chromatin is compact and transcriptionally repressed]
Histone acetylation occurs via what enzyme? What effect does this have on gene expression?
Histone Acetyl Transferase (HAT)
Promotes gene expression; acetylated chromatin is open and transcriptionally active
HATs act as ____________ while HDACs act as __________
Co-activators
Co-repressors
Dihydrotestosterone Estradiol Thyroid hormone Retinoic acid Vitamin D Cortisol Aldosterone Progesterone
The above serve as ligands in what type of signaling?
Nuclear receptor signaling
DNA methylation occurs when methyl groups are added to the DNA molecule, specifically to __________ and ___________, by ________ ____________ enzyme
Cytosine
Adenine
Methyl transferase
What is the effect of DNA methylation?
Changes the activity of the DNA segment without changing the sequence
Represses gene transcription when at a gene promoter, and is essential for normal development
What DNA alteration is associated with genomic imprinting, x-chromosome inactivation, repression of transposable elements, aging, and carcinogenesis?
DNA methylation
What is the effect of hypomethylation?
Chromosomal instability and/or loss of imprinting
Hypermethylation is associated with gene __________ and can arise secondary to gene ______________ (oncogene suppressor). It may be a target for ___________ therapy.
Promoters
Silencing
Epigenetic
Gene promoter CpG islands acquire abnormal ______________, thus inducing transcriptional _____________. This can be inherited by daughter cells following cell division.
Hypermethylation
Silencing
What form of each nucleotide is required for DNA replication?
dATP
dGTP
dCTP
dTTP
What does DNA polymerase require in order to begin processing?
A primer with a free 3’-OH
DNA-dependent DNA polymerase synthesizes new DNA in the ______ to ______ direction
5’ –> 3’
What does it mean for DNA replication to be bidirectional and semi-discontinuous?
Replication occurs in both directions from the origin of replication. The “leading” strand is synthesized continuously, while the “lagging” strand is synthesized discontinuously and forms Okazaki fragments
The building block of DNA is a deoxyribonucleotide, which is composed of 2-deoxyribose with a base (a purine or a pyrimidine) attached at the ____ position and a phosphate attached at the ____ position.
Remember that a base plus a (deoxy)ribose yields a _______________. Thus, a (deoxy)ribonucleotide is a (deoxy)ribonucleoside with 1-3 phosphate groups.
1’
5’
(Deoxy)ribonucleoside
How might nucleoside analogues act as inhibitors of DNA synthesis?
Because DNA synthesis involves the formation of 3’ –> 5’ phosphodiester bonds, nucleoside analogues that lack the 3’ OH group act as drugs that inhibit DNA replication. Such nucleosides need to be converted to dNTPs before they can act as inhibitors of DNA polymerase.
Arabinosylcytosine (Ara-C) is a nucleoside analogue drug used to treak Leukemia. How does it work?
Ara-C contains the sugar arabinose and is converted by animal cells into ara-CTP, a potent competitive inhibitor of DNA polymerase
Acyclovir is a nucleoside analogue drug used to treat herpesvirus. How does it work?
A herpesvirus encoded thymidine kinase catalyzies the initial phosphorylation of acyclovir, and the host’s enzymes convert the monophosphate into a triphosphate. Acyclovir lacks a 3’ OH group and therefore arrests viral DNA synthesis by acting as a chain terminator
AZT is used in HIV therapy as a nucleoside analogue drug. How does it work?
AZT is taken up by HIV infected cells and after activation is utilized by viral reverse transcriptase. AZT lacks a 3’ OH group and therefore arrests viral DNA synthesis by acting as a chain terminator
Describe the function of DNA helicase
6-subunit enzyme that unwinds DNA; binds and hydrolyzes ATP causing a conformational change that propels it like a rotary engine, prying apart the double helix
What DNA replication machinery binds cooperatively to exposed ssDNA and helps to stabilize it and prevents formation of hairpins?
Single-stranded binding protein (SSBP)
Fluoroquinolone is an antibiotic responsible for inhibiting what bacterial replication enzyme?
Topoisomerase (DNA gyrase)
What DNA replication enzyme is responsible for relieving overwound supercoils? What is this enzyme called in bacteria?
Topoisomerase
Called DNA gyrase in bacteria
What enzyme is responsible for synthesizing the RNA-DNA primer?
DNA polymerase alpha (in complex with primase)
What DNA replication enzyme is responsible for synthesizing the new DNA chain in the 5’ to 3’ direction? What is this called in prokaryotes?
DNA polymerase delta and epsilon
Called DNA polymerase III in prokaryotes
What DNA replication enzyme is responsible for removing RNA primers? What is this called in prokaryotes?
Flap endonuclease (FEN1) aka DNA polymerase I in prokaryotes
What DNA replication enzyme is responsible for sealing nicks?
DNA ligase
As the replication fork moves, it creates an unwinding problem for the parental helix. Every _____ bp replicated corresponds to one turn. DNA topoiosomerase is a reversible enzyme that breaks a _________ bond to relieve supercoiling and change superhelicity.
10
Phosphodiesterase
What are the benefits to low mutation rates in germ cells vs. somatic cells?
Germ Cells: low mutation rates maintain species
Somatic cells: low mutation rates avoid uncontrolled proliferation/cancer
There is 1 mistake in every 10^9 base pairs following DNA replication, which equals about 3 nucleotides changed for every cell division. Most repairs are corrected by proofreading and DNA repair. What enzyme is responsible for this proofreading?
DNA polymerase
What type of DNA damage-causing agent can come from the cell itself or from chemicals in the environment?
Reactive oxygen species
What is the result of UV radiation from the sun on DNA?
Can produce covalent linkage between 2 adjacent pyrimidines (T-T or C-T)
Aka pyrimidine dimers/thymine dimers
What are some potential results of ionizing radiation on DNA?
Strand breaks
40-60 chemical modifications of bases
DNA protein cross links (e.g, thymine-tyrosine crosslink)
What are some potential results of non-ionizing radiation on DNA?
Pyrimidine cyclobutane thymine dimer
6-4 covalent linkage between thymines
Depurination is the loss of a purine base from the DNA strand, what functional group takes its place?
OH
Why are methylated cytosine residues in CpG sites/islands problematic?
CpG sites are associated with inactive genes. The deamination of methyl-C produces T mismatched with G
Methylation of CpG islands stably silences genes. A special DNA glycosylase recognizes and removes the T. However, DNA repair is relatively ineffective in this case
What is the result of the deamination of Adenine?
Hypoxanthine (NH2 group is replaced by carbonyl)
What is the result of the deamination of guanine?
Xanthine (amino group replaced by carbonyl)
What is the result of the deamination of cytosine?
Uracil (amino group replaced by carbonyl)
How does benzo(a)pyrene, a compound produced in well-done grilled meats, affect your DNA?
Benzo(a)pyrene is converted by our own metabolic enzymes into BPDE (an epoxide), and forms an adduct with guanine
Benzo(a)pyrene is a pro-carcinogen and BPDE is a carcinogen
DNA damage can be caused by chemical agents. What type of agents are the following?
Nitrogen mustard
Cisplatin
Mitomycin C
Carmustine
Cross-linking agents
DNA damage can be caused by chemical agents. What type of agents are the following?
Dimethyl sulfate (DMS) Methyl methanesulfonate (MMS)
Alkylating agents
DNA damage can be caused by chemical agents. What type of agent is thalidomide?
Intercalating agent
Individuals with inherited mutations in one of the alleles of genes in the Mismatch excision repair (MER) complex (e.g., MSH2, MLH1) have an increased susceptibility to hereditary nonpolyposis colorectal cancers. How many mutations must occur for cancer to develop in this case?
An acquired mutation in the remaining good copy of the gene would render the MER system nonfunctional and allow tumor development
What disease is often the result of a defective nucleoside excision repair (NER) mechanism?
Xeroderma pigmentosum
Causes extremely sensitive skin in direct sunlight, increased risk of melanoma and squamous cell carcinoma. UV rays cause thymine dimers to form which are typically repaired by NER, but not in this case.
What disease typically results from a mutation affecting transcription-coupled DNA repair mechanisms?
Cockayne syndrome
Characterized by developmental/neurological delays, photosensitivity, and progeria. Also common are hearing loss and eye abnormalities. Death usually occus within 2 decades. Type B of cockayne is most common.
What type of genes are BRCA1 and BRCA2?
Tumor suppressor genes
Mutations in these cause a fivefold increase in a woman’s risk of developing breast and/or ovarian cancer before reaching menopause
What DNA repair mechanism fixes pyrimidine dimers and O^6-methylguanine via DNA photolyase and methylguanine methyltransferase?
Direct repair (enzymatic repair)
What type of DNA repair mechanism fixes single-base mismatches and nondistorting alterations (i.e., depurination) using enzymes like DNA glycosylase, AP endonuclease, AP lyase, DNA polymerase, and DNA ligase?
Base excision repair
What type of DNA repair mechanism fixes chemical adducts that distort DNA (e.g., pyrimidine dimers, BPDE-guanine adducts, and cisplatin adducts)?
Nucleotide excision repair (NER)
What type of DNA repair mechanism fixes mismatched bases in daughter strands via helicase/endonuclease, DNA polymerase, and DNA ligase?
Mismatch excision repair (MER)
What type of DNA damage/mutation causes hereditary nonpolyposis colorectal cancers?
Mismatched base in daughter strand
What type of DNA damage/mutation occurs in Xeroderma Pigmentosum?
Pyrimidine dimers
What type of mutation causes BRCA1/2 breast cancer?
Double-strand breaks, interstrand crosslinking
What type of DNA damage/mutation causes Cockayne syndrome?
Stalled RNA polymerase during transcription (not replication)
What type of DNA repair mechanism fills in and joins damaged ends, although some bases may be missing? Multiple proteins and enzymes are utilized including DNA ligase
Recombination repair (including nonhomologous end joining and homologous recombination)
What type of DNA damage is repaired by translesion synthesis?
Unrepaired thymine dimers or apurini AP sites
What genetic defect results in leukemia, lymphoma, gamma-ray sensitivity, and genome instability? What protein is affected?
Ataxia telangiectasia (AT)
Affects ATM protein, a protein kinase activated by double-stranded breaks
What genetic defect causes breast, ovarian, and prostate cancer? What process is affected?
BRCA2
Affects ability to repair by homologous recombination
What genetic defect causes congenital abnormalities, leukemia, and genome instability and affects the ability of DNA to undergo interstrand crosslink repair?
Fanconi anemia groups A-G
How are thymine dimers corrected by direct repair?
Photoreactivation (photolyase –> light)
How do DNA glycosylases perform base excision repair?
Altered base is detected by DNA glycosylases
Glycosylase removes base, AP endonuclease cuts phosphdiester bond, AP lyase removes deoxyribose phosphate
DNA polymerase beta replaces excised nucleotide
DNA ligase seals nick
MutS and MutL are proteins that operate in what DNA repair mechanism?
Mismatch excision repair
MutS binds while MutL scans for the nick and triggers degradation of the nicked strand
During mismatch excision repair, what distinguishes the new strand in E. Coli?
Methylation
What are the potential causes of double stranded breaks in DNA?
Ionizing radiation
Replication errors
Oxidizing agents
Other metabolites
If double stranded breaks are left unrepaired, chromosomes would break into smaller fragments and be lost. What repair process brings broken ends together and rejoins by DNA ligation, but one or more nucleotides will be lost?
Non-homologous end joining
What is transcription-coupled repair?
Best for sequences that urgently need repair. Cells can preferentially direct DNA repair to sequences that are being actively transcribed by linking RNA polymerase with DNA repair.
The RNA polymerase stalls at lesions and directs repair machinery there. Works with BER, NER, and others to repair damage.
With Cockayne’s syndrome, the RNA polymerase is permanently stalled at sites of damage in important genes
______ can acetylate nonhistone substrates such as transcriptionfactors and thus show promise in the treatment of several diseases like cancer, azheimers, and RA.
HATs
Cancer cells are very sensitive to inhibitors of ________ deacetylases. Inhibitors of _______, such as valproic acid and vorinostat are used as anticonvulsives and anticancer drugs
Lysine
HDACs
How does Rifampicin inhibit bacterial synthesis? What are the side effects?
Binds to the beta subunit of the RNA polymerase, thus preventing it from initiating translation
Good because human RNA polymerases are unaffected
Rifampicin upregulates hepatic cytochrome P450, thus increasing metabolism of other drugs like warfarin and contraceptives. May cause red color to urine, sweat, and tears
What nuclear receptor is activated by rifampicin?
PXR
Why is PXR important in terms of drug-drug interactions?
If providing a treatment that operates via PXR, it likely increases metabolism of any other drugs patient might be taking that also interacts with PXR
_____________ is a mechanism for regulating gene activity independent of DNA sequence that determines which genes will be on or off in different cell types, disease states, and physiological conditions.
Epigenetics
What type of enzymes are NAD+ dependent deacetylases?
Class III Sirtuins (Silent Information Regulator 2)
___________ is a small 76 amino acid protein that exists in all eukaryotic cells and is attached to _______ residues on target proteins, tagging them for destruction
Ubiquitin; Lysine
How many lysine residues does ubiquitin itself have, and which ones are best-characterized for the protein itself to be ubiquitinated?
7 total lysine residues
Lys 63 and 48
Ubiquitinated proteins are degraded by the __________
Proteosome
Lysines are what get ubiquitinated, but it is the _____ sequence specifically that gets attached to target proteins
GG (diglycine)
What are the 4 types of ubiquitination?
Monoubiquitination
Multi-monoubiquitination
Heterogenous polyubiquitination
Homogenous polyubiquitination
_____________ is a reversible PTM in which small ubiquitin like modifer proteins are covalently attached Lys residues in a process similar to _______________, which plays an important role in the normal functions of cells
Sumoylation
Ubiquitylation
