gochin's final material Flashcards
know thefunctions of DNA pol & other enzymes invovled in DNA synthesis
what happens in pre-replication events?
where does DNA replication begin and what occurs?
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− Pre-replication events – the nucleosome needs to be disassembled for replication to
occur
DNA replication starts with the origin of replication, which are AT rich sequence.
o Eukaryotes have multiple origins
− 20 DNA A binds to origin
o Uses ATP to unravel double strands by melting DNA
− Prepriming complex assists with unwinding of DNA and expands the region
o Breaks the hydrogen bonds between bases
o Uses ATP to separate strand
− Single stranded binding proteins (SSBs) binds to the single stranded DNA and stabilizes
the unwinding of DNA
o SSB proteins binds cooperative to the single stranded DNA and protects it from
degradation (from nucleases) & the two strands from reannealing
o Prepriming complex
− A replication bubble forms because it is unwinding in both directions and have
replication forks at both ends
know thefunctions of DNA pol & other enzymes invovled in DNA synthesis
what does DNA-A bind to?what occurs next?
DNA-A binds to native DNA helix
o Found in both eukaryotes and prokaryotes
but with diff code
o “melts” the DNA
− Helicase unwinds DNA
o 2 ATP is needed per A-T base pair
− As DNA unwinds, the strands ahead become
supercoiled, topoisomerase is used to relieve
the supercoil.
o Cleaves phosphodiester bond
− Topoisomerase I cleave the covalent bond of
one strand to relieve strain (no ATP required)
− Topoisomerase II cleave both strands to relieve
strain (requires ATP)
− Gyrase is a type of topoisomerase II but in
prokaryotes
o Antibiotics act on gyrase
know thefunctions of DNA pol & other enzymes invovled in DNA synthesis
explain what happens when inhibiting topoisomerase enzymes stopping DNA replication.
Inhibiting topoisomerase enzymes can stop DNA replication
o Topoisomerase I inhibitors are anti-cancer medications → prevents cancer cell
from replicating DNA
▪ Inability to relieve supercoil strain = replication is stopped.
− DNA polymerase epsilon comes in and synthesizes from 5’→3’
o Builds off from 3’ end of RNA primer
− Leading strand: the strand that is synthesized towards the replication fork.
o It is a continuous strand
− Now we will have an issue with the top strand since the parental strand is 3’ to 5’ in the
other direction (away from the expanding replication fork)
− Primase is initiates synthesis
o It is an RNA polymerase → does
not require a primer
− Primase binds to 3’ end of template and
generates a 10-nucleotide long RNA
primer.
o Reads from 3’ → 5’
o Synthesizes 5’ → 3’
− Uses NTPs and PPi is released
− The RNA primer has a 3’OH end where
DNA polymerase can bind to and make
DNA
know thefunctions of DNA pol & other enzymes invovled in DNA synthesis
what will primase do?what occurs after this and how does it arrive to making a complete daughter strand?
Primase will lay down a short RNA primer in the 5’ → 3’ direction
o Uses dNTPs releases PPi
− Since the parental strand is in the opposite direction (going from 3’ → 5’) of the
expanding replication fork, it is called the lagging strand.
− New primase needs to continue to lay down RNA primer when the replication fork opens
and new DNA polymerase gamma synthesizes away from the replication fork
− Okazaki fragments are short sequences of nucleotides which are synthesized
discontinuously
− RNA hydrolase binds and hydrolyzes
RNA, and removes the DNA primers
− DNA polymerase fills the games
with DNA
− DNA ligase will come in and ligate the
Okazaki fragments, connecting them
together, making a complete daughter
strand
o Requires ATP
know thefunctions of DNA pol & other enzymes invovled in DNA synthesis
how does replication of the 3 prime end occur?
Replication of 3’ ends
− 3’ ends of lagging strand will not be copied during replication
− Telomere shortening occurs with each replication cycle
− Cells reach a “hayflick” limit after which shortened telomeres trigger apoptosis
− Telomeres shorten with age
− Telomerase in germ cells regenerates at the ends of chromosomes
− Cancer cells are immortalized
− Telomerase uses telometric RNA that has complementary sequences to telomeric DNA
o Reverse transcriptase activity that acts on RNA to product DNA strand
o Conducts 5’ → 3’ synthesis
− Telomerase can translocate along the telomere of DNA and generate a new templated to
further elongate DNA.
o Extends telomeres by elongating 3’ end and then translocating back to the end and
cycle repeats
− Telomerase plays an important role in elongate and prevents ends from truncation
understand the mechanisms and key enzymes available for the repair of DNA damage. inclusing BER, NER, MMR, HR, and NHEJ and what type of damage is each process responsible for repairing
BER
In base excision repair, the damaged (or
abnormal) base is removed from its sugar
linkage and replaced.
− Abnormal bases such as alkylated or
deaminated bases, or nucleoside analogs
− DNA glycosylase breaks glycosidic bond and
removes the abnormal base
o An AP (apurinic) site is created
− AP endonuclease creates a nick in the
phosphodiester backbone where AP site is
created.
− DNA polymerase , DNA ligase, ATP are
used to add the complementary base pairings
to repair the site
______________________
NER
Most active in genes that are being actively transcribed
− Nucleotide excision repair (NER) removes large
distortions/lesions in DNA.
− Required for repairing UV damaged DNA (sun
damaged DNA)
− TFIIH (helicase) unwinds the DNA around the
damaged pyrimidine primer
− Endonuclease helicase nicks the phosphodiester
bond and the abnormal strands are removed
− DNA polymerase & , DNA ligase, and ATP
adds the complementary nucleotide sequence
− Individuals with XP are very sensitive,
demonstrate elevated incidence of skin
cancer, develop pigmented lesions upon sun
exposure, light sensitivity
___________________________
MMR
Mismatch Repair (MMR) occurs after DNA
replication as a “spellcheck” on its accuracy.
− Used to correct mismatches between abnormal
(non-Watson-Crick) base pairing.
− Uses BER and NER enzymes, as well as
specialized enzymes
− Mutations in MSH2 or MLH1 can lead to
predisposition for an inherited form of colon
cancer (HNPCC)
o Hereditary colon cancer
_____________________________
HR
Loss of heterogeneity can
occur which results in
deletion of a good allele
and progression to cancer
repair of double stranded breaks
removes chemotheraputically linked interstrand crosslinks
Homologous recombination repair (HRR) repairs double stranded breaks
− Involves processing the ends of the DNA double-strand break, homologous DNA pairing
and strand exchange, repair synthesis, and resolution of heteroduplex molecules
− Uses DNA sequence of homologous chromosome to repair a break in the other
chromosome
− Removes chemotherapeutic linked interstrand crosslinks
o Ex: Breast cancer (BRCA1, BRCA2 are enzymes of HR system)
o These genes recognize and recruit the correct enzymes for interstrand crosslinking
▪ Enzymes are responsible for recognizing breaks
__________________________________
NHEJ
Non-homologous end joining repairs double strand breaks that are caused by ionizing
radiation and chemical mutagens
o The break ends are directly ligated without the need for a homologous template
− A response to a DNA double stranded break caused by ionizing radiation or chemical
mutagens, such as intercalators
− Perfect integrity of bases at the repair junction may not be maintained
o Prone to errors
− Used by B and T cells for genetic rearrangements needed to produce their unique
immunoglobins and T-cell receptors (VDJ recombination)
− Defects in NHEJ → chromosomal translocation (and loss of genetic material?)
Defects in NHEJ may lead to
chromosomal translocation, where a gene
or portion of a gene is translocated from
one chromosome to another
− Ex: translocation from chr8 to chr14,
which causes a type of lymphoma of Bcells
− C-myc is translocated to another
chromosome and is transcribed much
more.
know the following DNA mutations and how they affect a DNA sequence and ultimate protein product:
Missense mutation
silent mutation
nonsense mutation
frameshift mutation
Codon triplet repeats
Missense mutations: a change in a base (point mutation) leads to a change in amino acid
________________
Silent mutation: a change in a base that results in the same amino acid → no change in
protein
o Often (not always) wobble base position and will result in the same amino acid
_____________________
Nonsense mutation: a change in a base that leads to a stop codon
o Leads to truncation of the protein at the C-terminus
________________________
Frameshift mutations: an insertion or deletion of 1 or 2 bases will shift the reading
frame and change the amino acid → changes protein
an mRNA sequence is decoded ins ets of three nucleotides (triplet) called a codon
know the various types of RNA in a cell and the polymerase responsible for their synthesis in eukaryocytes
RNA polymerase
− There are three different types required to synthesize different types of RNA
− RNA polymerase I: synthesizes rRNA (28S, 18S, 5.8S)
o rRNA makes ribosomes
− RNA polymerase II: synthesizes mRNA (hnRNA = heterogenous) and snRNA
− RNA polymerase III: synthesizes tRNA and 5S rRNA
− Mito: mitochondrial RNA
− There are a lot of transcription factors (proteins)
− RNA polymerase does not require a primer
− Prokaryotic RNA polymerase synthesizes all RNAs except primers for DNA synthesis
o Prokaryotic polymerase made up of core enzyme and sigma factor that recognizes
promoter
Pol I: pre-rRNA is modified and processed
by small nucleolar RNA (snoRNA),
splicing occurs. Gives 18S, 5.8S, and 28S
subunits
− Pol II: Nucleus makes small nuclear RNA
(snRNA) and pre-mRNA.
pre-mRNA us modified, spliced, edited,
and transported to cytoplasm as mature
mRNA
− Pol III: Synthesizes pre-tRNA, 5S, and
RNase P.
Pre-tRNA undergoes modification,
processing, splicing, and editing to make
tRNA
___________________
siRNA: exogenous, duplex RNA. Processed by RNase DICER, high homology to target
RNA. Knocks down mRNA
− miRNA: Regulates gene expression, endogenous, intergenic DNA, inhibition of gene
expression → reduces translation of mRNA
− stRNA: development and maintenance of organism
Describe the steps and factors invovled in protein synthesis on the ribosome and the consequences of inhibition at any step in the process.
Protein synthesis (1-4: preceding protein synthesis / 5-10: protein synthesis)
1. Dissociation of empty ribozyme (ending/beginning) – ribosomal subunit used over and
over again
2. Amino-acylation of tRNA and association with initiation factor
3. Formation of the pre-initiation complex
o Binding of mRNA and Met-tRNAiMet to the 40S ribosomal subunit (P site)
4. Formation of the 80S initiation complex
o GTP hydrolysis – GTP used to translocate tRNA
5. Elongation factor binding to tRNA
6. tRNA binding to the ribosome (A site)
7. Peptide bond formation
8. Translocation of ribosome along mRNA
o growing peptide attached to tRNA in the P site (GTP hydrolysis)
9. Release of empty tRNA from E site
o Will continue for each nucleotide (steps 5-9 repeats)
10. Termination at STOP codon with the aid of release factors
1st step: amino acid esterification
− Amino acid will associate with ATP
o Two phosphate groups cleaved
off and produces
pyrophosphatase
− AMP is on amino acid
− Generates a high energy bond which
will provide energy for peptide bond
formation.
o AMP-enzyme
2nd step: tRNA esterification
− CCA (tRNA) associates with the amino
acid by AARS.
− Amino acyl tRNA synthetase (AARS):
an enzyme that attaches an amino acid
to its tRNA by adding to 3’ end
o the first adapter in protein
sequence
o AMP released
− Results in amino-acyl tRNA (charged
tRNA)
Amino-acyl tRNA synthetase is the 1st adaptor = recognizes a specific tRNA moelcule and
amino acid
o Binding pocket is specific for amino acid
o Binding pocket is specific for anticodon base triplet
− tRNA is the 2nd adaptor = connecting the codon in mRNA with anticodon in tRNA
o Ensures the correct amino acid will be incorporated into the sequence based on the
tRNA sequence
o allows for wobble base pairing
3. Formation of pre-initiation complex (PIC)
− Ribosomes (rRNA + protein)
o Eukaryotes: 40S (small) + 60S (large) = 80S ribosome
o Prokaryotes: 30S (small) + 50S (large) = 70S ribosome
Initiation factors / eIFs assist in forming
the PIC
− mRNA sequence is brought into the
small subunit
o CAP recognition plays a role in
the small subunit binding
− CAP recognition and translocation of
tRNA to the P-site
− eIF-2 will bring the initiator tRNA
(met)
o GTP is required for bringing in
Met-tRNAiMet
− Antibiotics acts on prokaryotes
ribosomal subunit (30S)
− When antibiotics inhibits mRNA binding
by acting on prokaryotic 30S subunit,
the formation of the pre-initiation
complex would be prevented.
Large subunit binds to pre-initiation complex and forms complete ribosome.
− eIF-2 and other initiation factors are released
− When antibiotics inhibit the prokaryotic 50S subunit, then the formation of the
initiation complex (complete ribosome) is inhibited
− In prokaryotes, the initiator tRNA is loaded with formyl-met-tRNA initiator
5 and 6. Chain elongation
− Elongation factors: carry out chain elongation
− Initiation factors: brings in met-tRNA
− EF1alpha brings new tRNA with amino acid into
the A site (translocating)
o GTP is used to deliver amino acid
o Hydrolysis of GTP to GDP will
inactivate the transcription factor
− When antibiotics act on the EF-Tu in
prokaryotes, then elongation will be inhibited
because tRNA is not being translocated.
− EF-1 in eukaryotes
− EF-Tu in prokaryotes
7. Peptide bond formation
− P = peptidyl tRNA
− A= new amino acid
− Peptidyl transferase: catalyzes the addition
of amino acid to the growing peptide chain
o Activity component is in 28s rRNA
(23S in prokaryotes) – carrying out
the transfer activity
− Makes peptide bonds
− When antibiotics act on the 23S rRNA in
prokaryotes, it will inhibit the transferase
activity and peptide bonds will be
inhibited
8. Ribosome translocation
− EF-2 uses GTP to mediate
translocation of tRNA along the
ribosome
o GTP is hydrolyzed to GDP,
which provides the energy
needed to move the tRNA from
the A→P site.
− A site is empty and a new tRNA can
come in.
− When antibiotics act on EF-G in
prokaryotes, it inhibits ribosome
translocation by preventing
translocation/shifting of tRNA.
o Prokaryotes 50S subunit
− EF-2 in eukaryotes
− EF-G in prokaryotes
− Diptheria inactivates eEF-2 by
preventing recycling to GTP
bound state.
− Toxins replace GTP with ADPribose group, which inactivates
eEF-2
− Protein synthesis is inhibited at
the ribosome translocation step
9. Release of tRNA from E Site
10. Termination
− Termination occurs when a stop codon is recognized.
o UGA, UAG, UAA
− RF1 and RF2 (release factor) bind to the A site at the termination codon and delivers a
water molecule
o Uses GTP
− Complete peptide chain is transferred to water molecule
− RFs are similar to tRNA, which is why it can bind onto the A site to cause hydrolysis
Ribosome recycling factor (RRF) binds at the A site and causes all the components to
release and dissociation of the ribosomal subunits
− Overall, protein synthesis is very energetic – high energy cost to cells
1. Initiation
o Addition of met-tRNAimet to P site → 1 GTP
o Addition of mRNA → 1 ATP
2. Elongation
o Addition of aa~tRNA to A site → 1 GTP
o Peptide bond formation (aa~tRNA bound)
o Ribosomal translocation → 1 GTP
3. Termination → at least 1 GTP
know how gene experssion is affected by histone modification and DNA methylation, and the enzymes involved.
Regulation at the level of DNA
− Regulation at the DNA level includes:
o Chromatin remodeling
▪ DNA methylation
▪ Modulation of histone association
o DNA amplification
DNA methylation causes gene silencing
by blocking transcription
− CpG islands regulate gene expression
through transcriptional silencing of
corresponding gene
o regulatory regions in
housekeeping genes
o C-phosphodiester bond-G
− DNA methyl transferase transfer
methyl group to DNA
o Uses S-adenosyl methionine
(SAM) as the methyl donor
− miRNA involved in DNA methylation
− Reduces transcription → gene silenced
Methylated DNA is said to be imprinted.
− Methylation is implicated in the imprinting of genomic DNA
o Heritable, retaining memory of parental origin
− Methylation patterns can be acquired – changed throughout life
− Expression of gene is changed, NOT the sequence
DNA methylation
− Methylated DNA binding proteins also recruit histone modifying enzymes
o A mechanism for chromatin remodeling
o Heterochromatin tightly condense and euchromatin loosely condensed due to
remodeling
− Histone DNA association is enhanced
by the fact that DNA is negative, and
histones are positive
o Histone is positive due to
arginine and lysine
o Electrostatic interaction
− Histone acetyltransferase (HAT):
enzymes that add acetyl groups onto
lysine residues on histones
o Recruited by transcription
factors
o Makes euchromatin
− Histone deacetylase (HDAC): enzyme
that removes acetyl group from histones
o Recruited by methylated DNA
base pair
− Acetylation will reduce lysine strength
of charge, making it neutral
know the location of basal promoter and gene-specific receptor elements relative to a gene, and the proteins that bind to them. Know the examples of each. Understand cis-acting vs. trans-acting regulators.
Regulation at the level of transcription
1. Cis-acting regulatory sequences are regions of non-coding DNA, which regulate
transcription of nearby genes
o promoter, enhance, repressor
Structural gene: made of exons,
introns, and promoter on the
coding portion of the gene
− Regulatory sequences: interact
with molecules that interact with
promoter and alter level of
transcription
o Basal promoters
o Silencer/enhancers
Basal promoter: binds to transcription factors
o Usually upstream of RNA polymerase II
o Ex: TATA Box
− Silencer/enhancer: gene-specific regulatory DNA sequence
o Can be anywhere on sequence
o Ex: Hormone response element, CpG island, cAMP response element
2. Trans-acting molecules regulate (or modify) the expression of distant genes
− Usually protein factors that bind to cis-acting sequence
− Includes: basal transcription factors, miRNA
o TFIIE, hormone receptors, CREB
− Responds to small molecules for transcription
o Hormones, cAMP
Combinatorial control of
transcription
− This is the basal promoter.
Basal transcription complex
and regulatory molecules can
regulate transcription
− RNA polymerase binds to
TFII to start transcription
Upstream of the promoter sequence, there is a regulatory sequence where a
transcriptional activator can bind and recruit other proteins, such as HATs
o Associates with basal transcription complex
Hormonal control with hormonal
receptor
− The GRE is a sequence of DNA that
is cis-acting regulatory molecule.
− Glucocorticoid receptor is transacing. It binds to DNA and binds
only when it is bound to its own
hormones
− Recruits coactivators and can bind to
basal transcription complex and
initiate transcription.
o Glucocorticoid will be
associated with transcription
of specific genes
