test1 cards week1b Flashcards
reverse transcriptase
catalyzes the synthesis of DNA from RNA template
retroviral genetic material
is RNA, they use reverse transcriptase to make DNA from their RNA
Telomerase
maintains ends of chromosomes (telomeres), uses an RNA template to do this, so it has reverse transcriptase activity
end of replication problem
leading strand can be synthesized until the very end, but the lagging strand cannot (read the end of the DNA replication handout)
telomere lengthening
cancer cells can do this to make cells immortal
DNA is the only macromolecule that is repaired
DNA is irreplaceable, so 100’s of genes are required for DNA repair
base los s (depurination, depyrimidination) repair
BER
deamination of A, C, or G
BER
UV 2+2 dimers
direct reversal or NER
base alkylation repair
direct reversal, BER or MMR
base oxidation by ROS repair
BER or MMR
bulges in helix caused by insertion or deletion of nucleotides repair
NER
Bulky chemical adducts causing bulges repair
NER
mismatch errors repair
MMR
closslinking repair
SSBR or NER
Strand break repair
direct reversal, SSBR or DSBR
Staller DNA replication forks
DSBR
deamination
can cause pt mutation: eg 5-methylcytosine loses NH3 to make thymine
base alkylation
can cause pt mutation; due to H-bond changes will replicate differently
reversal of a specific single stranded DNA break
direct reversal by DNA ligase
reversal of UV T-T or T-C dimers
direct reversal (photolyase)
reversal of base alkylation
direct reversal: O6-meG methyltransferase (MGMT)
repairs base damage that does not distort DNA
Base Excision repair
repairs base damage that does distort the DNA
Nucleotide excision repair
nucleotide excision repair
distorded DNA is recognized by protein endonuclease also cleaves phosphodiester bonds; helicase unwinds; exonuclease removes nucleotide; polymerase installs complimentary nucleotide; ligase closes the gap
Global genome NER
recognizes distorting genome lesions in any region
transcription coupled NER
recognizes lesions in regions that are being transcribed
mutations in genes that mediate NER
lead to diseases such as Cockayne synd, xeroderma pigmentosum, trichothiodystrophy
base excision repair
altered base specific glycosylases recognize and remove; DNA pol replaces; ligase fixes nicks
mismatch repair
MutS and MutH (MSH and MLH in mammals) recognize mismatches, endonuclease cleaves phosphodiester bonds, exonuclease removes nucleotide, helicase unwinds, DNA Pol replaces, ligase fixes nicks
MMR detection of new strands
nascient lagging strand has transient 5’ DNA ends from okazaki frags; nascient leading strand is marked by transient ribonucleotides
mutations in MMR
cause hereditary non-polyposis colorectal cancer
lesion bypass
used when too much DNA damage for NER, BER, or MMR to handle. Allows for bypass or error prone pol’s with loosened specificity to continue thru damaged region. They lack a proofreading 3’ to 5’ exonuclease. HIGHLY MUTAGENIC
Double strand breaks
repaired by non-homologous end joining or homologous recombination
non-homologous end joining
often innacurate, can result in insertion or deletion
homologous recombination
used homologous template DNA; very accurate
single strand breaks
caused by: collapsed DNA replication, stalled transcription, and PARP (poly(ADP-ribosylation)
DNA DAMAGE CHECKPOINT
pauses cell cycle to allow for DNA repair; signalling pathways using protein kinases ATM and ATR. Or can lead to apoptosis
deficiencies in DNA damage checkpoint
genomic instability and malignant conversion
DNA control elements
TATA box, promoter, proximal elements, enhancers
TATA box
initiator sequence usually 25-35 bps upstream of the start site, determines site of transcription initiation and directs binding ofRNA polII
promotor proximal elements
200 bps upstream of TSS, ~20bps long, help regulate transcription, and can be bound by factors in a celltype specific manner
enhancers
contain multible control elements each 8-20 bps long, thus an enhancer can be 100-200 bps long; usually 200 to 10’s of kilo bases upstream OR downstream from the promoter
b-thalassemia
an inherited anemia due to deficient production of b-globin proteinby erythroid cells. Occurs due to different types of mutations one of which can be in the b-globin promoter, thus reducing the amount of b-globin mRNA produced
Hemophilia B Leyden
X-linked clotting disorder, affected males have 1% of normal factor IX until puberty. The factor IX gene has mutations in the promoter - preventing binding of the approriate transcriptional activators. At puberty, androgen receptors can bind to the active site and increase transcription
Fragile X syndrome
FMR1 gene facilitates methylation of cytosine residues in CpG islands. FMR1 gene normally has 6-50 CGG repeats in the 5’ region, affected males (1 in ~1500) have >200 CGG repeats and leads to increased silencing of the FMR1 gene. This results in mental retardation, dysmorphic facial features and post pubertal macroorchidism
transcription activators and repressors
Proteins encoded by one gene that act on other genes to regulate their transcription. Can therefore diffuse around the nucleus and affect transcription of numerous genes. Can either activate or repress transcription.
two classes of transcription activators and repressors
Sequence-specific DNA binding proteins and Co-factors
Sequence-specific DNA binding proteins
bind to promoter or enhancer elements (DNA control elements) in their target genes to regulate transcription. The elements they bind to are usually 6-8 base pairs long. Usually bind DNA by inserting their _-helices into the major groove of DNA, making contacts between the amino acid side chains of the protein and the bases in the DNA
co-factors
Do not bind directly to the DNA elements but rather bind to sequence-specific DNA binding proteins and affect transcription through this contact.
two major domains of ss DNA binding proteins
DNA binding domains - confer sequence specificity; activation domain - very unstructured, medaite protein-protein interactions that recruis general transcription machinery and/or co-activators that modify chromatin
classification of sequence specific transcription factors
classified by their DNA binding domains: homeodomain proteins (helix-turn-helix); Zinc-finger proteins; basic leucine zipper proteins; basic helix-loop-helix (bHLH)
Craniosynostosis
characterized by the premature closure of one or more sutures in the skull and affects 1/3000 infant; one varient is causes by a mutation of the homeodomain protein MSX2
Androgen insensitivity syndrome (AIS)-
AIS includes feminization or undermasculinization of the external genitalia at birth, abnormal secondary sexual development in puberty, and infertility. Caused by mutations in either the DNA binding domain or the ligand binding domain of the androgen receptor (a zinc finger DNA binding protein)
Waardenburg Syndrome type II
characterized by deafness, pigmentation anomalies of the eyes, and other pigmentation defects (hair, skin). Mutations in the microphthalmia-associated transcription factor (MITF) gene (which encodes a bHLH DNA binding protein) are observed in 15-20% of the patients.
dimerization of seq specific DNA binding factors
The Zinc finger, bZIP, and bHLH can all form heterodimers. If each monomer of the heterodimer has a different DNA binding specificity, the formation of heterodimers will increase the number of potential sequences to which that family of sequence specific transcription factors can bind. Combinatorial Control
How do transcriptional activators or repressors, once bound to DNA control elements, stimulate transcription?
- Activators and Repressors regulate assembly of initiation complexes and rate of initiation of transcription 2.Activators and Repressors regulate changes in chromatin structure influencing the ability of general transcription factors to bind promoters
what are the two classes of chromatin remodeling factors?
- DNA-dependent ATPases (SWI/SNF)- disrupt histone octamers and DNA. 2. Factors that reversibly modify histones through acetylation (HATs and HDACs)
nucleasome
a core of histone proteins around which DNA is wound
histone N- termini
rich with lysine residues, which can be reversibly modified by acetylation, phosphorylation, methylation, and ubiquitination. Acetylation is associated with gene control.
Activators and Repressors can recruit:
either histone acetyltransferases (HATS) or deacetylases (HDACs).
Histone acetyltransferases (HATS)-
a co-activator; acetylates lysine; allows for binding of specific transcription factors - thus serves as a ‘code’ to recruit different factors that will then affect the transcriptional site
Histone deacetylases (HDACs)-
a co repressor; de-acetylates
trans-acting transcription factors
can switch from repressors or activators by recruiting HAT’s or HDAC’s
activators
eg. CBP or p300; recruit HATs
repressors
recruit HDACs
some diseases involved in histone acetylation
Leukemia, epithelial cancers, Rubenstein-taybi synd, polysomy X, Fragile X syndrome
Rubinstein-Taybi Syndrome
rare genetic multisystem disorder (affects 1/125,000); Characterized by growth retardation, mental retardation, craniofacial dysmorphism, abnormally broad thumbs and great toes. Results from mutations in one copy of the CREB binding protein (CBP) gene. CBP is an essential transcriptional coactivator for many different transcription factors and is a histone acetyltransferase.
Leukemia-
A hematopoietic malignancy. Are generally the result of chromosomal translocations leading to gain of function fusion proteins- some of which involve fusions of transcriptional regulators with HATs or HDACs, altering the activity of the regulators .
euchromatin
where genes reside; more accessible form of chromatin
heterochromatin
always repressed because inaccessible found near centromeres, telomeres, and internal chromosome positions, so inaccessible
Constitutive Heterochromatin
is always heterochromatin and contains satellite DNA. Example - centromeric DNA
Facultative Heterochromatin
can change to euchromatin, depending on the cell type or developmental stage, and is enriched in LINE sequences. Example - X-inactivation
How are the sequence-specific DNA binding proteins regulated?
- The conformation of the DNA-binding protein can be altered by ligand binding 2. Entry into the nucleus can be regulated 3. The amount of transcription factor in the cell can be regulated 4. DNA binding can be regulated 5. Phosphorylation of the DNA-binding protein can alter various properties including protein degradation, recruitment of co-activators, and DNA binding
Overview of Basic Principles of Transcriptional Regulation
see slide 54 - FORD
The nuclear receptor family of Zinc finger transcription factors work by binding to:
Steroid hormones
ligang binding can affect:
dimerization of receptor, recruitment of coactivators/repressors, and translocation into the nucleus. Examples include estrogen receptor activation and glucocorticoid receptor activation
Estrogen receptor binds DNA as a homodimer
through a pair of Zn fingers
estrogen binds to estrogen receptor which causes
its dimerization
tamoxifen
estrogen agonist: competitively binds to ER and prevents recruitment of HAT co-factors
regulating entry into the nucleus example
NF-kB is normally in the cytoplasm when bound to IkB.In response to a variety of stimuli, I_B can be phosphorylated, targeting it for degradation. NF_B is then released and moves to the nucleus, where it turns on a number of target genes, including those involved in inflammation
aspirin
works in part by inhibiting the phosphorylation, and thus degradation of IkB. This prevents the translocation of NFkB to the nucleus, thus inhibiting the transcription of genes involved in the inflammatory response.
dephosphorylation of cytoplasmic NF-AT:
High intracellular calcium activates calcineuin’s phosphatase activity
activated calcineurian phosphatase:
dephosphorylates cytoplasmic NF-AT. This exposes the nuclear localization sequence, allowing NF-AT to enter the nucleus where it affects transcription of genes involved in the immune response and in heart function.
immunosuppresants cyclosporin and FK506 mode of action
inhibit calcineurin, thereby inhibiting NF-AT action.
Extracellular regulators stimulate Wnt to:
ultimately destibilize the Axin-APC-GSK3 complex which is needed to phosphorylate beta catenin for degradation. The resulting increase in beta catenin allows for some to move to the nucleus, where it interacts with the TCF family of transcription factors and promotes the expression of Wnt responsive genes.
APC mutations
increase cellular beta catenin - has implications in colon cancer
p53 downregulation
p53- is downregulated by binding to the MDM2 protein which not only masks its activation domain, but also targets it for destruction by the ubiquitin-proteasome pathway. *Note importance of p53 in human cancers.
Example of regulating DNA binding:
Id proteins- The Id family members negatively regulate DNA binding by heterodimerizing with other HLH proteins through their HLH domains, but preventing DNA binding due to their lack of a basic domain.
Example of how phosphorylation affects activity of trans-acting factors:
phosphorylation of CREB (cyclic AMP response element-binding protein) promotes transcription be allowing recruit ment of CBP/PolII - leading to transcriptional activation of the gene
