Exam 2 Flashcards
4 characteristics of genetic material/DNA
- needs to have the information
- transmissionable
- replication
- variation, must be capable of change and account for change
Exaplin experiment that explained transformation
Bacteria that was deathly was killed and put into a mouse with alive bad bacteria but the mouse still died.
took DNA from the dead bacteria somehow
transforming principle
Exaplain how they knew that DNA is genetic material
During the mouse experiment with S/R bacteria, only the extract with DNA transformed R bacteria into S bacteria
Also, DNAse made is so the transformation didn’t happen but RNAase didnt kill the DNA
4 levels of nucleic acid structures
- Nucleotides
- Single stranded DNA
- Double helix
- 3d structure
the builiding blocks of DNA is
nucleotides
_ are linked _ to make DNA strands
nucleotides are linked covalently
Nucleotide components
- phosphate group
- pentose sugar (ribose or deoxyribose)
- nitrogenous base
nucleoside components
base + sugar
DNA strands consist of _ bonds
ester bonds that link nucleotides together
ester bonds are the P=O bonds in the phos group
phsophediester linkage
nucleotides 5’ to 3’ linkages
nucleotides 5’ to 3’ linkages are called
phosphodiester linkages
in a DNA strans all sugars are orientated….
in the same direction
what forms the backbone of the DNA strand
phosphate and sugar molecules
the _ project from the backbone of DNA
bases
DNA stores info in…
the base sequence
the _ are the _ ring bases
- purines - double ring
- pyrimidines - single ring
purine and pyramidine bases
list them
purine: A,G
pyrimidines: T, U, C
difference between DNA and RNA
DNA sugar has a hydrogen not an OH on the 2nd carbon
Linus Pauling used _ to discover the _ helix
ball and stick models to discover the alpha helix
Franklin used _ to see the _ of DNA
X-ray diffraction to see the molecular patterns of DNA
showed that DNA is helical, more than one strand, and has around 10 base pairs per turn
Chargaff analyzed _ to discover _
the base concentrations of DNA from several specices to discover Chargaff’s rule
A = T
C = G
base pairing
Watson and crick used _ to discover _
ball and stick models to discover the double helix structure of DNA
Key features of DNA double helix
- clockwise, right handed helix
- bases in opposite strands hydrogen bond AT/CG
- 2 strands are antiparallel
- 10 base pairs in each turn of the helix
base stacking
in DNA, flat parts of bases face each other and stabalize by the hydrophobic effect
A is bonded to T with
2 H bonds
C is bonded to G with
3 H bonds
important for protein binding to DNA are
the major and minor groove, which you need to recognize the bases to see
Z dna characteristics
- left handed
- bases tilted alot
- backbone is zigzag
RNA double helix features
- right handed
- 11 to 12 base pairs per turn
Types of RNA secondary structures
- Bulge loop
- internal loop
- multibrached loop
- stem loop
draw these
factors contributing to tertiary structure of RNA
- base pairing and base stacking within RNA
- interactions with ions, molecules, etc
features of bacterial chromosome
- one origin of replication
- genes
- intergenic locations
- repetitive sequences
nucleoid
place where bacterial chromosome is found
* not bound by a membrane
microdomains
loop domains to compact bacterial DNA
bacteria use _ to form micro and macrodomains
nucleoid associated proteins (NAPs)
NAPs function
nucleoid associated proteins
- fascilitate chromosome compaction
- bend DNA for it to bind
- chromosome segregation fascilitation
- gene regulations
ways DNA in bacteria becomes compact
- NAPs
- supercoiling
_ supercoils are good for
negative supercoils food for transcription and compacting DNA
how does negative supercoiling help dna transcription
it creates tension that is released by strand seperation
the control of supercoiling is done by
- DNA gyrase
- DNA topoisomerase 1
DNA gyrase function
- uses ATP to make negative supercoils
- relaxes supercoils
- untangles DNA
increase transcription, very active
DNA topoisomerase 1 function
relaxing negative supercoils
complex eukaryotes have _ genes with many _
longer genes with many introns
3 types of DNA sequences required for eukaryotic replication
- origins of replication
- centromeres
- telomeres
eukaryotic chromosome key features
- linear
- occurs in sets
- very long with many genes
- multiple origins of replication
- centromere
- telomeres
- repeatitive sequences
repeatitive sequences are usually found near
centromeric and telomeric regions
sequence complexity
the number of times a particular base sequence appears in the genome
three main types of repeative sequences
listed
- unique or non repeatitive
- modereatly repeatitive
- highly repeatitive
unique sequences
includes protein encoding genes and intergenic regions
* most of the genome
moderately repeatitive sequences
- genes for rRNA and histones
- regulate gene expression and translation
- transposable elements
highly repeatitive sequences
- fairly short
- some interspersed throughout genome
- some are clustered together in arrays
most of eukaryotic genomes is
repeatitive DNA
transposition
the integration of small segments of DNA to a new location
two general types of transposition pathways
brief description
- simple transposition - TE moves to a new site using a transposase
- retrotransposition - TE moves to a new site using an RNA intermediate
TE = transposable elements
proliferation of retrotransposons happens with…
reverse transcriptase and integrase
OR
target site primed reverse transcription
in simple transposition and retrotransposition, how many copies of the TE are there
simple: 1 copy (just moves)
retro: 2 (make copy then move)
direct repeats and why they exists
- repeats that flank the TE and same direction
- exist so that transposase can do a staggered cut when inserting gene
inverted repeats
function and defin
- repeats that are opposite directions (palindroms)
- exist so the transposes can cut TE out
insertation element has
- direct repeat
- inverted repeat
- transposase gene
simple transposon has
- direct repeat
- inverted repeat
- transposase gene
- gene for another thing like antibiotic resistance
LTR retrotranspoons contain
- direct repeat
- long terminal repeat
- reverse transcriptase gene
- integrase gene
non LTR retrotransposon contains
- direct repeat
- reverse transcriptase or endonuclease gene
no LTR and no integrase
transposable elements are considered to be _ when _
autonomous elements when they contain all info for transposition
steps of simple transposition
- transposon binds to inverted repeats
- transposon dimerizes and creates a loop
- transposase cleaves outside inverted repeats
- transposase cleaves target DNA at staggered sites
- the transposable element is inserted
- direct repeats are made
draw it out!
simple transposition can increase _ by _
increase copy number by inserting ahead of the replication fork
LTR retrotransposition process
- transcription of TE with reverse transcriptase multiple times
- integrase places into target DNA sites
can proliferate in multiple sites
non LTR retrotransposition
- retrotranspoon is transcribed
- one strand of target DNA is cleaved by endonuclease
- the retrotranspoon RNA with a poly-A tail attaches to the cut DNA like a primer using
- reverse transcriptase copies RNA into DNA to finish the DNA cut part by adding the TE with transcription
- endonuclease cuts other strand
- RNA gets yeeted off, new TE DNA added into the target sequence
- DNA polymerase fills in gaps
abundance of TEs is most in
complex eukaryotic multicellular organisms
selfish DNA theory
TEs exist bc they can
hypothesis for why TEs exist
- selfish DNA theory
- TE offer some sort of advantage
- TE increase geneic variablility
- exon shuffling (may lead to genes with more diverse functions)
nucleosome structure
double stranded DNA segmenet wrapped around octomer of histones
* 2 copies of each histone type: H1, H2A, H2B, H3
one histone is composed of
- globular domain
- an amino terminal tail
- many positively charged amino acids (Lys, Arg)
histones and dna binding
histones have many posistive AAs which bind to the negative phosphate groups in the DNA backbone
H1 histone
- linker histone
- less tightly bound
- helps to organize adjacent nucleosomes
Strings on a bead model was tested by
using DNase 1 to cut DNA into fragments, hypothetically linker region should cut which they were
30 nm fiber
at low salt concentrations, H1 remains bound and the beads of nucleosome come closer together, shortens DNA by alot
3 ways loop domains are formed
to further compact DNA
- 2 CCCTC binding factor (CTCF) binds to DNA then bind to each other
- SMC protein forms a dimer than wraps itself around DNA and makes a loop
- or both of the above happens
drawing chap 10 slide 88
each chromosome can be found in
chromosome territory
the predominant structure in the chromosome territories is
loop domains
2 levels of compation in interphase
list
- heterochromatin
- euchromatin
hetrochromatin
- tightly compact region
- no transcription
- loop domains v compact
euchromatin
- less compact regions
- transcriptionally active
- 30 nm fiber loop make loops
two types of hetrochromatin
list and properties
- consitutive: regions that are permenantly hetrochromatic, usually have lots of repeats, around centromere and telomere
- facultative: can be both euchromatin or hetrochromatin
process of entering metaphase
- DNA double helix
- wrapping DNA to make nucleosomes
- formation of zigzag into 30 nm fibers
- 30nm fibers anchor to proteins to make loop domains
- loop domains come closer to one another
- chromosome
condensin
role
- condensin 2 enters nucleus during interphase and helps with condensing
- 1 stays in cytoplasm
- at end of prophase both help to compact loops
cohesin
role
- found along length of chromatid and holds chromatids together
- degreaded when prophase begins
Due to the _ DNA can be replicated to produce two double helices with the identical base sequences?
Due to the AT/GC rule
What are the expected results for the Meselson and Stahl experiment after 4 generations (i.e, 4 rounds of DNA replication in the presence of light nitrogen)? Note: during generation zero, the DNA is all heavy, and subsequent generations only make light DNA.
1/8 half heavy
7/8 light
How many replication forks are formed at an origin of replication?
2
Primase is needed during DNA replication because DNA polymerase is not able to
begin synthesis on a bare template strand.
In the leading strand, DNA is made in the direction _ the replication fork and is made as _
toward, one continuous strand
During proofreading, DNA polymerase
cuts out a mismatch by digesting DNA in the 3’ to 5’ direction.
Which ends of DNA are extended by telomerases?
3’ end
DNA replication relies on
AT/GC rule
3 different models proposed for the replication of DNA
list and explain
- conservative model: both parental strands stay together
- semiconservative model: parental strands split
- dispersive model: random parental and daughter DNA segments
if conservative model was true, the Mendelson and Stahl experiments 1st and second generation (start with heavy DNA)
- generation 1: 50% heavy, 50% light
- generation 2: 25% heavy, 75% light
semi conservative model, Mendelson and Stahl experiments 1st and second generation (start with heavy DNA)
- generation 1: all half heavy
- gen 2: 50% half heavy, 50% light
if dispersive model was true, Mendelson and Stahl experiments 1st and second generation (start with heavy DNA)
gen 1: all half heavy
gen 2: all 1/4 heavy
DNA synthesis begins at
origin of replication
oriC 3 types of important DNA sequences
list
- DnaA boxes
- AT rich regions
- GATC methylation sites
DnaA boxes
sites for the binding of DnaA protein
part of oriC
AT-rich regions
sites where DNA strands seperate
part of oriC
GATC methylation sites
sites that make sure DNA replication doesnt start again too soon
part of oriC
events that occur at oriC
- DnaA proteins bind to DnaA boxes and to each other
- DNA bends
- strands seperate at AT rich regions
- DnaB/helicase binds to the origin to further seperate DNA strands
- repliation occurs in both direction
DNA helicase is combosed of
6 subunits
DNA helicase travels along DNA in the _ direction
5’ to 3’
how do GATC sites regulate replication
Dam methylates the A on both strands, DNA replication only starts of fully methylated DNA and the daughter cells are not, so replication doesn’t restart too soon
DNA helicase seperates strands by _ and in turn generates a _
by breaking H bonds between them and generating a positive supercoiling ahead of each replication fork
DNA gyrase travels ahead of the helicase and
alliviates positive supercoils
_ bind to DNA strands to keep them apart
SS binding proteins
after DNA strands are seperated,
RNA primers are synthesized by primase
the leading strand has _ primers
1
the lagging strand has _ primers
multiple
_ are responsible for synthesizing DNA
DNA polymerase
the lagging strand leads _ from fork
away
DNA pol 1-5 in e coli functions
- normal replication: 1 & 3
- DNA repair: 2, 4, 5
DNA pol 3
- responsible for most DNA replication
- alpha unit catalyzes bond formation
- DNA pol haloenzyme is 10 units
DNA pol 1
struc and func
- composed of a single polypeptide
- removes the RNA primers and replaces them with DNA
features/problems of DNA polymerase
2
- cannot initiate replication on bare strand (needs primer)
- can only attach 5’ to 3’ but strands are anti parallel (fragments)
DNA pol 1 uses _ activity to _
- endonuclease activity to digest RNA primers
- polymerase activity to replace it with DNA
after DNA pol fills DNA in primer spots…
DNA ligase comes in to form covalent bonds
DNA ligase
function
forms covalent bonds between primer and DNA and fragments
DNA helicase and primase form a complex _ in order to _ , this complex also forms _ with two DNA holoenzymes
DNA helicase and primase form a primosome to coordinate, the primosome comes with a holenzyme to form a replisome
how does the replisome avoid hoping
creates a loop of where the next okazaki fragment will be synthesized
what stops movement of replication forks in e coli
tus bond to ter, termination sequences
T1 stops
counterclockwise forks
T2 stops
clockwise forks
catenanes
- intertwined DNA molecules after replication
- seperated by topoisomerase 2
DNA pol catalyzes the formation of _ between
a ester bond between P of new base and 3’OH of old base
2 Ps are released
in formation of an ester bond, _ is release
PPi
2 P s
processive feature of DNA pol 3 is due to
DNA pol 3 stays attached to template because of the beta subunit forming a clamp around the template DNA
in absense of the beta subunit…
DNA pol 3 would fall off the DNA template, very inefficient
DNA replication has a high degree of
fidelity (accuracy)
why is fidelity high in DNA replication
- stability of base pairing
- structure of DNA polymerase at active site (incorrect pair causes weird shape)
- proofreading of DNA polymerase
proofreading is done with
3’ to 5’ endonuclease activity of the DNA pol
* digests new strand until the mismatch is found
why is eukaryotic DNA replication more complex
- larger linear chromosomes
- tightly packed
- cell cycle highly regulated
eukaryotes have long linear chromosomes, so
they require more origins of replication so that replication is done quickly
characteristics of origin sites in simple eukar
- lots of A and T
- consensus sequence
ARS elements
origins of replication in simple eukaryotes
in eurkar, replication begins with
- the assembly of preplication complex (preRC)
the preRC includes
list
- origin recognition complex
- MCM helicase
origin replication complex
struc and func
- six subunit complex
- first initiator of eukar DNA replication
binding of MCM completes
DNA replication licensing
eukar strand seperation done by
MCM helicase
explain how primers are removed in eukary
- Polymerase δ starts the removal of primers by making an Okazaki fragment longer and creating a flap in the adjacent fragment.
- Flap endonucleases then remove the flap.
- This process repeats until the DNA primer is completely removed
DNA polymerases in mammals and which DNA they act on
- nuclear DNA: alpha, delta and epsilon
- mitochondrial DNA: gamma
DNA pol alpha is the only to
associate with primase
what makes primer in eukary
DNA pol alpha and primase complex synthesizes a short RNA-DNA hybrid primer
10 RNA nucleotides followed by 20-30 DNA
what makes primer in eukary
DNA pol alpha and primase complex synthesizes a short RNA-DNA hybrid primer
10 RNA nucleotides followed by 20-30 DNA
polymerase switch
- exchange of DNA pol alpha for DNA pol epsilon or delta
- required for elongation
_ is needed for elongation in eukar
exchange of DNA pol alpha for DNA pol epsilon or delta
DNA pol epsilon is used for
elongation of leading strand
DNA pol delta is used for
elongation of lagging strand
_ are involved in replication of damaged DNA in eukar and they can _
translesion-replicating polymerases, they can synthesize a complmentary strand over an abnormal region
if the flap is too long in RNA primer removal in eukar,
DNA2 helicase/nuclease cuts the flap shorter
why do eukar chromosomes have telomers
because 3’ end of DNA cannot be replicated due to the lack of a primer, a primer cannot be added to nothing
telomeres consist of
- 3’ repeatitive tandem overhands
- several G
- many T
if there were no telomeres…
linear chromosomes would get shorter every round of DNA replication
telomerase creates _ that is
adds DNA sequences to the 3’ end that is RNA that is complmentary to the DNA in the telomeric repeat
telomerase steps
- binding to 3’ overhang region
- polymerization: synthesis of a repeat
- translocation: telomerase moves the repeat to the right and makes another repeat
- the completion of this primer can be used now
chap 11 page 72
telomerase tend to
shorten in actively dividing cells
when telomeres are short
cells lose their ability to divide
cancer cells carry
mutations that increase activity of telomerase in order to prevent telomere shortening and continue dividing
