MFM - Lecture 1 + 2
Terms in this set (105)
DNA Replication and Regulation Lecture 1
DNA replication occurs during the S phase of the cell cycle
S phase lies between the two gap phases (G1 and G2) and takes 6-8hr to complete
- Produces 2 double helices from original DNA
- Each new DNA helix is identical in nucleotide sequence to the parental DNA double helix
- DNA serves as a template for its own duplication
- DNA is duplicated in a semiconservative manner
Steps in DNA replication
Origin of replication
Particular sequence of DNA at which replication is initiated
Typically is composed of an AT rich sequence
In eukaryotes, there are multiple origins of replication along chromosomes
Establishment of Replication Origion
ORC complex binds to origin (origin recognition complex)
MCM complex binds to the ORC complex
Helicase binds to the ORC/MCM complex and begins separating the strands to form a small bubble
Single Strand Binding Proteins (SSBs), such as RPA, bind to the exposed single strands and hold them apart, keeping the double strand from reforming
Replication forks form at each end of the origin of replications and progress in opposite directions
Separation of the parental DNA strands and unwinding of the helix ahead of the replication fork
Separates the DNA strands and unwinds the parental duplex
Uses the energy of ATP hydrolysis
Enzyme that breaks phosphodiester bonds and rejoins them
Relieves the supercoiling of DNA caused by unwinding
Single Strand Binding Proteins
such as RPA, bind to the exposed single strands and hold them apart, keeping the double strand from reforming
DNA polymerases can only add nucleotides to the 3' end
Because the double strand is antiparallel, the polymerases need to move in opposite directions to make the two new strands
Each strand is made in a different way
- leading strand
- lagging strand
Polymerase moves along the 3' to 5' strand, assembling the new strand 5' to 3'
Polymerase has to go in the opposite direction due to orientation of the template
In order to keep up with the replication fork, the polymerase must replicate the lagging strand in short segments (Okazaki fragments)
Strand of template DNA that is oriented so that the replication fork moves along it in a 5' to 3' manner
The lagging strand is replicated in a discontinuous fashion
Produces small DNA fragments, later joined by DNA ligase to form a continuous new strand
Synthesizes a short RNA primer with a 3' OH group
Removes the RNA primer from 5' ends of Okazaki fragments
Processes the 5' end of Okazaki fragments to allow for ligation
Fills in gap left behind by the removal of the RNA primer
Joins the 5'-phosphate end of one new DNA fragment to the adjacent 3'-hydroxyl end of the next
Enzyme that is responsible for incorporation of nucleotides into the newly synthesized DNA strand
Pols α, δ, ε
DNA replication and repair
Pols κ, η, ξ, ι "Bypass polymerases"
Bypass areas of damaged DNA
Lack 5' exonuclease activity
Binding of DNA polymerase to the DNA
Clamp-loading protein binds to DNA
RCF (Replication Factor C)
- clamp loader protein
Clamp-loading protein assembles clamp
("Sliding clamp") around DNA
DNA polymerase associates with the clamp
Clamps increase the processivity of the polymerase
Allow polymerase to stay attached to the DNA for a longer period of time
PCNA (proliferating cell nuclear antigen)
- proteins that form the "sliding clamp"
In Eukaryotes, chromosomes have multiple replication forks
When these replication forks meet, they resolve each other and terminate replication
Replication of Chromosome Ends - Telomeres
Found at the ends of linear chromosomes
Repeating sequence of bases (TTAGGG), repeated thousands of times
Replicated by telomerase
- RNA-dependent DNA polymerase
- Contains both proteins and RNA template
- RNA template has complimentary sequence to repeating sequence in the telomere
A section of the telomere is lost during each cycle of replication at the 5' end of the lagging strand due to lack of DNA upstream to ligate to
Inefficient telomerase activity can prevent the addition of the nucleotide repeats at the end of the chromosome
Contributes to the aging process
As an organism ages, the telomeres in its cells become progressively shorter
Permanent changes in the DNA
Can be inherited if it occurs in germ cells
Potential consequences of DNA mutations:
Prevent a gene from functioning properly
Affect the product of a gene (protein)
No affect on the gene or gene product
Causes of Mutations
Does not break the phosphodiester backbone
Results in loss of nitrogen base
When the replication machinery
encounters a missing purine on
the template strand, it can skip to
the next complete nucleotide,
producing a nucleotide deletion
in the newly synthesized strand
Does not break phosphodiester
Conversion of a cytosine to uracil
The DNA replication machinery
inserts an adenine when it
encounters a uracil on the
Excites water in the cell, leading to the generation of hydroxyl radicals
Hydroxyl radicals react with DNA, altering the structure of the bases or cleaving the DNA strands (double strand breaks)
Cause formation of pyrimidine dimers
Mutations that result in the formation of covalent bonds between carbons in adjacent thymines (thymine dimers)
Aromatic polycyclic hydrocarbon
When oxidized by cytochrome P450, forms bulky adducts with guanine residues in DNA
Types of DNA Mutations
A mutation that causes a change in a single nucleotide in the DNA sequence
Results in a codon that codes for a different amino acid
Results in a premature stop codon
Extra nucleotide is added
Can change the reading frame and result in a frameshift mutation
Loss of a nucleotide
Can change the reading frame and result in a frameshift mutation
Results from slippage during DNA replication
The newly synthesized strand dissociates from the template strand, a kink is formed, and repeat
sequences allow the new strand to re-anneal in
the wrong location, creating a duplication of that
The greater the number of repeats, the more
likely that a disease will occur or the severity
of a disease will increase
Caused by trinucleotide expansion
in the gene encoding the protein
<28 CAG (glutamine)
>40 CAG repeats
Gross chromosomal rearrangements resulting from breaks in chromosomes
The free ends of the DNA at the break point reseal with the free ends of a different broken chromosome
Frequently observed in cancer cells
Some hereditary diseases are associated with chromosomal translocations
How are DNA mutations repaired
Two major types of DNA damage that need repaired
Single nucleotide defects
Nucleotide excision repair
Base excision repair
Double stranded breaks in DNA
Non-homologous end joining
Excision repair (is used when damage occurs to nucleotides in one strand)
Nucleotide excision repair - Specific repair endonucleases cleave the abnormal chain and remove the damaged region
The gap is then filled by a DNA polymerase that adds deoxyribonucleotides, one at a time, to the 3'-end of the cleaved DNA, using the intact complementary DNA strand as a template
The newly synthesized segment is joined to the 5'-end of the remainder of the original DNA strand by a DNA ligase
*** (IMPORTANT) Used for the removal of thymine dimers and bulky adducts
base excision repair
DNA glycosylase cleaves the N-glycosidic bond that joins the damaged base to deoxyribose
The sugar-phosphate backbone of the DNA now lacks a base at this site
An endonuclease cleaves the sugar-phosphate strand at this site
DNA polymerase fills in the gap
DNA ligase joins the newly synthesized segment to the original DNA strand
*** (IMPORTANT) Used to correct spontaneous mutations introduced during replication (due to deaminations and depurinations)
Defect in nucleotide excision repair due to mutations in one of the XP genes (XPA, XPB, XPC, XPD)
Inability to remove UV-damaged bases results in the accumulation of mutations
Individuals with this disease are highly sensitive to sunlight
Develop multiple pigmented growths on the skin
High risk of skin cancer
Half of children with XP develop skin cancer by the age of 10
Active during DNA replication when an incorrect but normal base is incorporated into the growing chain
Mismatched bases do not form normal Watson-Crick base pairs
The mismatch is recognized by the mismatch repair enzyme complex
The mismatch repair enzyme complex removesa segment of the newly synthesized DNA that includes the mismatched bases
DNA polymerase and ligase repair the gap
DNA MISMATCHES ARE DUE TO REPLICATION ERROR
Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndrome)
Due to mutations in either MSH2 or MLH1
Cells are unable to repair nucleotide mismatches
Afflicted individuals are at increased risk of developing cancers in a number of tissues including:
Non-Homologous End Joining
Non-homologous end joining (NHEJ)
In somatic cells, this is the most common mechanism for repairing double strand breaks
Does not require a homologous chromosome as a template
Following a double strand break, nucleases process the broken ends to form blunt ends
- During this process, some nucleotides are lost
MAY INTRODUCE MUTATIONS
The ends are then brought together by a specialized group of enzymes and rejoined by DNA ligase
"Quick and dirty" DNA repair mechanism
COMMONLY USED BY CELLS IN THE G1 PHASE
Error-free method for repairing double strand breaks
Requires the presence of a homologous chromosome to be used as a template
Commonly used for repair of newly replicated DNA
1) Two homologous chromosomes become aligned
2) A nuclease generates single-stranded ends at
the break by chewing back one of the
3) One of the single strands then invades the
homologous DNA duplex by forming base pairs
with its complementary strand. A significant
number of bases must pair to produce a branch
point where one strand from each duplex
4) The invading strand is elongated by DNA
polymerase, using the complementary strand as
5) The branch point migrates as the base pairs
holding together the duplexes break, and new
6) Additional DNA synthesis and ligation complete the
SINCE SISTER CHROMATIDS (IDENTICAL COPIES) ARE REQUIRED, THIS PROCESS MUST OCCUR AFTER REPLICATION (G2, M)
Afflicted individuals have ataxia and telangiectasia
Due to a mutation in ATM
Normally, ATM is recruited to sites of double stranded breaks by binding to the DNA break recognition proteins
Individuals with this disease have an impaired DNA damage response
They have defective NHEJ and homologous recombination repair
They are susceptible to agents that cause double stranded breaks in DNA
Transscription and Transcription Regulation Lecture 2
Prokaryotic Transcription - Prokaryotic Transcriptional Unit
Prokaryotes are polycistronic (many of their genes are found in operons, in one transcriptional unit).
Spacing of the -10 and -35 sequences in the promoter is important for sigma factor binding.
Core Enzyme: 5 subunits (α2ββ'ω)
Holoenzyme: core enzyme + σ factor
Sigma factor (σ) have higher affinity for the DNA and will bind over the -35 and -10 sequence.
Transcription in Bacteria
1. Holoenzyme (RNA polymerase + sigma factor) bind promoter sequence.
2. RNA polymerase unwinds DNA
3. Transcription starts
4. After first 10 bases, sigma factor is release
5. RNA polymerase change form slightly and transcription continues.
6. RNA polymerase reaches termination sequence.
7. Hairpin structure disrupts polymerase and dissociates with DNA.
Requires 2 sequences in RNA
1. GC-rich hairpin
2. 8 uridines
The formation of the hairpin strains the conformation of the polymerase. The weak connection between the uridines and the templates are broken.
Rho protein binds a cytosine-rich region and travels the RNA in a 5' to 3' direction (chasing polymerase). When the polymerase stalls, the rho protein will disrupt the RNA and polymerase association.
Inhibitor of prokaryotic RNA polymerase: Rifampin
Inhibits transcription by binding to the β subunit of the RNA polymerase (stops phosphodiester bond formation in the RNA).
Used for the treatment of tuberculosis (TB).
Inhibitor of prokaryotic RNA polymerase: Fidaxomicin
Binds the SIGMA FACTOR of RNA polymerase and inhibits transcription.
Only effective in a small class of bacteria (narrow spectrum) due to each bacteria having a slightly different sigma factor.
Used to treat C. DIFFICILE infection (symptoms: watery diarrhea, abdominal pain, fever, dehydration, blood or pus in stool).
Very expensive $$$
RNA pol I-III
RNA pol I
most rRNA genes
RNA pol II
PROTEIN CODING GENES - miRNA genes, genes for some small NRA
RNA pol III
55 rRNA genes
genes for many small RNAs
Eukaryotic Transcriptional Unit
Eukaryotic transcription factors
general transcription factors TFII
Regulatory transcription factors
general transcription factors TFII
always used by RNA polymerase II to initiate transcription.
Regulatory transcription factors
proteins involved for the recruitment of general transcription factors and RNA polymerase to the promoter. Specific to genes or subset of genes.
TFIID binds TATA box (through TBP)
TFIIB and TFIIA bind DNA near TATA box
RNA Polymerase II is recruited to Promoter with TFIIs (E,F,H)
TFIIH opens helix (requires ATP) at start site and phosphorylates Polymerase II tail (CTD).
Phosphorylation leads to releasing TFIIs and progression to elongation stage of transcription.
Inhibitors of eukaryotic RNA polymerase II: α-amanitin
Binds to RNA polymerase II (also RNA pol III but less effectively) and inhibits transcription.
Found in the death cap mushroom Amanita phalloides
Latency stage of 6-24 hours followed by gastrointestinal symptoms (vomiting, diarrhea, and cramps), recovery period prior to kidney and liver failure, eventually death
Transcription Elongation and Termination linked to RNA processing
Eukaryotic mRNA is capped, spliced and tagged with a poly-A tail during the transcription process.
5' cap and poly-A tail provides protection from decay, signals "complete RNA", aids in export from nucleus, recruitment of the ribosome for translation.
Removal of introns allows for alternative splicing
RNA processing is regulated by the CTD of Polymerase II
The complexes involved in RNA processing are recruited to the CTD of polymerase II. This brings them in proximity to the newly synthesized mRNA.
Phosphorylation of the CTD by TFIIH regulates RNA processing.
5' Methylguanosine Cap
Capping occurs after ~25 nucleotides of RNA are synthesized. Facilitated by enzymes bound to the tail (CTD) of Polymerase II
Removal of 1 phosphate from the 5' of pre-mRNA
Addition of guanosine monophosphate (GMP) in a 5' to 5' linkage
Addition of methyl group to the guanosine
snRNA: small nuclear RNA, small (less than 200 bases) RNA used to identify sequence for pre-mRNA splicing. Five different snRNAs (U1, U2, U4, U5, and U6).
snRNPs: complex of snRNA and associated proteins that facilitate the splicing.
RNP: ribonucleoprotein, a complex of RNA and protein
Spliceosome: the complex and dynamic machine that is responsible for RNA splicing. Composed of snRNPs that associate with the mRNA at various times in the process. Utilizes ATP to complete splicing process.
Binding of snRNPs
snRNPs bind the introns in pre-mRNA through base pairing.
Donor site: 5' end of intron, AGGU sequence
Acceptor site: 3' end of intron, AG sequence
formation of the lariat structure
The 5' donor site will attach to an adenosine residue in the intron forming a lariat structure. The free end of the exon then attaches to the 3' acceptor site.
mutations in splice sequences
Mutations in the sequences required for splicing can lead to the inclusion or exclusion of sequence. The change in mRNA sequence will interfere with the production of the correct protein.
Example of mutations in splice sites: β0-thalassemia
Mutation destroys or create splice sites.
Little or no β-chain of hemoglobin is made, reduces levels of hemoglobin
Symptoms: if only one copy of β-globin gene is affect then symptoms include mild microcytic anemia, bone marrow erythroid hyperplasia, and occasionally
hepatosplenomegaly. Symptoms are more severe with both copies of gene effected
Eukaryotic transcription termination: Poly-A-Tail
As Polymerase II is transcribing RNA, the sequence AAUAAA will be made.
Cleavage Stimulation Factor F (CstF) and Cleavage Polyadenylation Specificity Factor (CPSF) will transfer from the CTD of Polymerase II to the RNA sequence.
This recruits other proteins leading to
1. Cleavage of the 3' end of RNA
2. Addition of the poly-A tail by Poly-A Polymerase (PAP)
3. Binding of the poly-A-binding protein (PABP) to the poly-A tail
Regulation of Transcription
Prokaryote Gene Regulation
Since transcription and translation are coupled reactions in prokaryotes, most gene expression is regulated at the level of transcription.
There are two main means of gene regulation in prokaryotes
- Interchangeable RNA polymerase subunits
- Genetic Switches
Altering RNA polymerase subunits in prokaryotes
Different sigma factors have different affinity for various promoter sequences. This allows specific sigma factors to turn on a subset of genes.
Increases transcription by enhancing the binding of the polymerase to the promoter or opening the helix.
Turns genes on
Binds the operator, a sequence upstream of transcription, blocks access to promoter for RNA polymerase.
Turns genes off
Eukaryotic regulation of transcription
Promote/ prevents assembly of transcription initiation complex.
1. Modification of chromatin
2. DNA methylation
3. Gene-specific transcription factors
Transcription factors cannot bind the promoter if DNA is tightly wrapped in nucleosomes.
4 mechanisms of chromatin modifications
1 .Covalent histone modifications
2. Nucleosome removal
3. Nucleosome replacement
4. Nucleosome remodeling
Lysine amino acids on the histone tails have a positive charge. When they become ACETYLATED they loose that charge and loosen their interaction with the negative charge of DNA.
turns on gene expression
Disorder caused by mutations in EP300 and CREBBP, genes encoding enzymes responsible for histone acetylation.
Symptoms include: intellectual disability, distinct facial features, microcephaly, broad thumbs and toes, short stature, predisposition to cancer, eye abnormalities, heart and kidney defects.
Used as a drug for epilepsy and bipolar disorder (Divalproex).
Recently discovered to be an inhibitor of histone deacetylases (HDACs)
Currently being studied as a chemotherapeutic. Cancer cells have abnormal gene expression. Inhibition of deacetylation allows for expression of genes that should be turned off.
Inhibit histone deacetylases (HDACs) leading to increased histone acetylation (or lack of removing the acetyl groups) and an increase in gene expression.
Binds zinc molecules from active site of HDAC enzymes and inhibiting the enzyme.
Used to treat T-cell lymphoma
Nucleosome Removal: Removing nucleosome to expose sequence for transcription (TATA box)
Replacing core histones with different variants that have a specific function
Sliding nucleosome to expose sequence for transcription (TATA box)
Coloboma (developmental eye defect), Heart defects, Atresia of the choanae, Retardation of growth, Genital underdevelopment due to hypogonadotropic
hypogonadism, Ear abnormalilties
Mutation in the CHD7 gene which encodes chromodomain helicase DNA binding protein 7. The CHD7 protein regulates gene expression by chromatin remodeling
GC-rich sequences (GC-islands) exist in the promoter region.
Cytosine is methylated to produce 5-methylcytosine
Methylation of cytosine reduces the ability of transcription factors to bind.
Inherited disorder (X-linked dominant) caused by a loss-of-function (lof) mutation in the MECP2 gene which encodes methyl cytosine binding protein 2 (MeCP2).
MeCP2 is expressed in many organs but concentrated in brain, lung, and spleen
MeCP2 is a transcriptional repressor for genes involved in brain function and development.
Symptoms include intellectual disability, repetitive hand movements and developmental regression.
Fragile X Syndrome
Dynamic mutations which increase the CGG repeats in the promoter of the FMR1 gene (encodes protein FMRP)
Leads to increase in methylation at the promoter, decrease transcription of the gene
The more CGG repeats, the decrease in mRNA produced.
X-linked recessive: more common in males
Symptoms include: intellectual disability, ADD-like symptoms, prominent jaw, large ears, long and narrow face, and enlarged testicles (after puberty)
Gene-specific Transcription Factors
Positive and negative control
Contain DNA-binding motifs
Used to bind other transcription factors and recruit (or prevent the recruitment) of RNA polymerase.
Transcription Factors in development
Gene regulation by transcription factors is essential during early development.
Many mutations in transcriptions factors lead to a diverse
set of disorders (heart defects, CNS defects, limb defects)
Focused on in detail during the Developmental Genetics Lecture.
Nuclear Receptors as transcription factors
Nuclear receptors are transcription factors that require the binding of a ligand for activation.
The receptor will bind a specific sequence in the promoter regulating the expression of the gene.
Mechanism for signaling for steroid receptor family
The hydrophobic steroid crosses the plasma membrane into the cytoplasm.
The hormone binds the receptor displacing the heat-shock promoter.
The receptors bind each other forming dimers and travel to the nucleus
The dimer complex acts on the hormone response element in the promoter while binding to other transcription factors.
Promotes the expression of specific genes
Nuclear Receptors as Drug targets
Selective estrogen receptor modulators (SERMs) bind the estrogen receptor preventing estrogen from binding.
Example: Tamoxifen is used to treat receptor-positive breast cancer.