Functions and Dysfunction in Genomic Regulation Flashcards
Central dogma of biology?
DNA to RNA to Protein
How can RNA become DNA?
reverse transcriptase, such as in HIV
How is DNA organized?
into chromosomes
How is DNA structured?
Helix due to hydrogen bonding
Anti Parallel due to hydrogen bonding
Purines?
A and G
Pyramidines?
C, T ,U
How much of the DNA genome is used for coding? For how many proteins?
1.5%, 100000
What form are mitotic chromosomes in during interphase?
Condensed, avoid damage to chromosomes
What is the Histone octamer and what does it make?
-basic unit of chromosome packing
“beads on a string”
Whats so significant about AA that make up histone?
- 20% Lysine or Arginine
- Attracted to negatively charged DNA backbone
- Targets of post-translational modifications (PTMs)
what is Histone deacetylase?
represses gene expression by compacting (winding) the chromatin
activated by nuclear receptors
what is Histone Acetyltransferase?
activates gene expression by acetylating and unwinding chromatin
What is euchromatin?
lightly packed DNA
Often but not always under active transcription
92% of human genome is euchromatic
what is Heterochromatin?
Contains few active genes
tightly bound
Position effect: activity of a gene depends on relative position of chromosome
Where do acetylation / methylation /ubiquitination occur?
N-terminus of Histone proteins
What is methylation? what and how does it do it? what is the significance?
Represses gene transcription when at gene promoter
Uses cytosine and adenine via methyl transferase enzyme
Associated with: genomic imprinting, X-chromosome inactivation, repression of transposable elements, Aging, carcinogenesis
Altering DNA methylation -> important component of cancer development
What is the significance of CpG Islands?
Gene promoter regions
can acquire abnormal hypermethylation leading to transcriptional silencing
this can be inherited by daughter cells following cell division, if in germ cells
can give rise to oncogene suppressor silencing, might be a target for epigenetic therapy
What is hypomethylation?
chromosomal instability, loss of imprinting
Phosphorylation, what is it and what does it do?
negative phosphate on positively charged histones would decrease the affinity histones have for negatively charged DNA, allowing for increased expression.
What does DNA replication need to occur? How is it replicated?
DNA polymerase requires a primer with a free 3’ -OH to begin processing
Replication is Semiconservative
Bi-directional replication
Synthesized 5’ to 3’
Helicase?
Unzips DNA Helix
Topoisomerase? what does it do
Relieves overwound supercoils
Breaks phosphodiester bond
Blocks cell cycle
Generate single/double stranded breaks
Harms integrity of genome
Leads to apoptosis and cancer cell death
Targeted in cancer therapy
What are the inhibitor of topoisomerase?
Topoisomerase I inhibitor:
Irinotecan is inhibited
Used for colorectal cancer
TOPOisomerase- II Inhibitor
Etoposide & Anthracyclines are inhibited
Anthracyclines cause cardiotoxicity
- Doxorubicin
- Daunorubicin
Etoposide causes secondary Leukemias.
DNA ligase?
Seals nicks
DNA polymerases?
Synthesize new DNA chain in the 5’ -> 3’ direction
Single-stranded binding protein?
Binds the single-stranded DNA that
has been separated
What Nucleoside analog inhibitors? what do they do? whats their significance?
LACK 3’-OH GROUP, inhibit DNA replication
Nucleosides analogs need to be converted to dNTPs before they can act as inhibitors of DNA polymerase
Examples: ara-C, Acyclovir, AZT
what is ara-C?
Treatment of leukemia
Ara-C contains the sugar arabinose, which is converted to ara-CTP by animal cells
Potent competitive inhibitor of DNA polymerase
What is Acyclovir?
Treatment of herpes/shingles lesions
A Herpesvirus-encoded thymidine kinase phosphorylates Acyclovir and the host’s enzymes convert the monophosphate to triphosphate
What is AZT?
treatment for HIV
Drug is taken up by HIV-infected cells and uses the viral RVTase to arrest viral DNA synthesis by acting as chain terminators.
What does Ionizing radiation do?
causes strand breaks
causes DNA protein cross links
40 to 60 chemically distinct base damages
What does non ionizing radiation do?
produces a covalent link between two adjacent pyrimidines (T-T or C-T)
makes pyramidine dimers
causes Basal and squamous cell carcinoma
what/how do spontaneous mutations in DNA happen?
spontaneously
DNA replication errors, base deletion or substitution
What is depurination?
(A or G) is removed from the nucleotide via hydrolysis of N-glycosidic bond between the base and the deoxyribose group
make an apurinic (AP) or abasic site
What is deamination? how and what happens?
amino group of purine or pyrimidine base is hydrolyzed
Adenine -> hypoxanthine
Guanine -> xanthine
Cytosine -> uracil
Forms unnatural deoxyuridine (dU)
what are intercalcating agents?
A chemical that can insert itself between the stacked bases at the center of the DNA double helix, possibly causing a frameshift mutation
thalidomide!!
What did thalidomide do!
Children born with short “flipper-like” appendages
horrible birth defects
Why are methylated cytosine residues in CpG islands bad?
methylation of CpG islands stably silences genes (cancer/DNA repair genes)
Loss of amine of methyl-c produces T mismatched with G
what is Benzo(a)pyrene?
Makes BPDE, carcinogen
What are cross linking agents?
exogenous or endogenous agents react with two nucleotides of DNA, forming a covalent linkage between them
These adducts interfere with cellular metabolism, such as DNA replication and transcription, triggering cell death
Nitrogen mustard
Cisplatin
Mitomycin C
etc
Alkylating Agents, what are they and what do they do?
substance that causes replacement of hydrogen by an alkyl group especially in a biologically important molecule; specifically : one with mutagenic activity that inhibits cell division and growth and is used to treat some cancers.
DDS,MMS
What is direct repair?
uses DNA photolyase & methylguanine methyltransferase
enzymatic
what is base excision repair?
DNA glycolases, AP endonuclease, AP lyases (of DNA polymerase), DNA polymerase Beta, DNA ligase
Nucleotide Excision Repair, why is it significant?
NER protein complex, DNA polymerase Beta, DNA ligase
Similar to MER but uses different proteins in process, but outcome is the same
Deficiency in NER leads to xeroderma pigmentosum
Mismatch Excision Repair (MER)- ?
MER complex, helicase/endonuclease, DNA polymerase Beta, DNA ligase
what is recombination repair?
Nonhomologous end joining- Damaged ends filled in and joined; some base pairs may be missing. Multiples proteins used including DNA ligase
Homologous recombination- Exonuclease, DNA polymerase, MER system
Deficiency leads to BRCA1/2 Breast cancer risk
Transcription-Coupled Repair (TCR)?
Deficiency leads to cokayne syndrome
stalling transcription
Translation Synthesis
Uses DNA polymerase
what does direct repair fix?
Pyrimidine Dimers & O6-methylguanine
what does Base Excision Repair fix?
Single base mismatches, nondistoring alterations (depurination)
what does Nucleotide Excision Repair fix?
Chemical adducts that distort DNA (pyrimidine dimers)
what does Mismatch Excision Repair fix?
mismatched base in daughter strand
what does Recombination Repair fix?
double-strand breaks, interstrand cross-linking
what does Transcription-Coupled Repair fix?
stalled RNA polymerase during transcription
what does Translation Synthesis fix?
unpaired thymine dimers or apurini AP sites
what is Xeroderma Pigmentosum?
photosensitivity, prone to developing melanomas and SCC
UV light causes cyclobutane thymine dimers to form in DNA
With defective XP proteins in NER complex show disease
Hereditary nonpolyposis colorectal cancer?
Inherited mutations in one of the alleles in MER complex have increased risk to HNPCC
Cockayne syndrome?
Defect in transcription-coupled repair
growth retardation, skeletal abnormalities, sensitivity to sunlight
RNA polymerase is permanently stalled at sites of damage in important genes
BRCA associated breast cancer?
Mutations in these genes cause 5x increase in woman’s risk in getting breast/ovarian cancer pre-menopause
Also associated with increased risk of cervical, uterine, pancreatic and colon cancer in women
*Recombination repair
What is Epigenetics?
study of heritable changes in gene expression (active versus inactive genes) that do not involve changes to the underlying DNA sequence
Histone Deacetylases (HDACs), what do they do?
enzymes that remove acetyl groups from lysines on core histones and Transcription factors, repressing genes
Cancer cells sensitive to inhibitors of such lysine deacetylases
HDAC Inhibitors, what are they and what do they do?
Vorinostat, Entinostat, valproic acid
Anticonvulsants and anticancer drugs
Rifampicin, what does it do?
inhibits bacterial RNA synthesis
Binds to B subunit of RNA polymerase,preventing it from initiating translation
Treats tuberculosis
Side Effects: upregulation of liver cytochrome p-450, which increases metabolisms of other drugs and hormones
*Red urine, sweat and tears
What are the CpG sites?
Left: sites every 1/10 nucleotide, gene promoter
Right: every 1/100 nucleotides, commonly methylated
what is the effect of methylation on gene transcription?
Gene suppression
what is the effect of sumoylation on gene transcription?
Similar to ubiquitination
what is the effect of Ubiquitination on gene transcription?
Ubiquitin attaches to lysine residues to signal to proteins for cell degradation (in proteasome; signal dependent)
what is the effect of Histone acetylation/deacetylation on gene transcription?
Acetylation- gene activation
Deacetylation- gene suppression
what is the effect of Phosphorylation on gene transcription?
Signals for degradation via proteasome
