Structure, Function and Replication of DNA Flashcards
What does DNA contain?
genes
What are genes? (2)
units of biological information and units of inheritence
Is DNA a polymer or a monomer?
polymer
What is a polymer?
a long strand (compound made up of a long chain of identical or similar units)
what are the monomers of DNA called?
nucleotides
What is a bacteriophage?
a virus that infects bacteria and reproduces inside of it using its viral DNA to hijack the host bacterial DNA
What is phage T2?
A bacteriophage
What is the genetic material of phage T2?
DNA
Describe the method of Hershey and Chase’s 1952 experiment? (3)
1) they infected bacteria with phage T2 labelled with 32P in DNA and 35S in protein
2) they separated the phage (protein) coat and the infected bacteria
3) they isolated progeny phage
What did Hershey and Chase find in 1952? (2)
The phage contained mostly 35S label and the infected bacteria mostly 32P. When progeny (offspring) of phage was isolated, they found 30% 32P and less than 1% of 35S (basically none)
What do Hershey and Chase’s 1952 findings show?
- The DNA, not the viral protein enter the bacteria
- the DNA of the parent phages becomes part of the progeny phage
How many parts does each DNA nucleotide have?
3
What are the 3 parts of each DNA nucleotide?
- A sugar
- base
- phosphate group
Why is the sugar 2’ deoxyribose (2 prime)?
the 2nd carbon of the sugar ring doesn’t have an oxygen attached (deoxy-)
How does the deoxygenation of the 2nd carbon in the sugar affect stability?
It increases it
How many phosphates in the phosphate group of DNA?
3
what are the names of the 3 phosphate groups of DNA?
alpha (a), Beta (B) and Gamma (y)
Where is the alpha phosphate positioned on the nucleotide?
closest to the sugar
Where is the beta phosphate positioned on the nucleotide?
between the alpha and gamma phosphates
where is the gamma phosphate positioned on the nucleotide?
furthest from the sugar
How many bases are in DNA?
4
What are the four bases?
Adenine, Guanine, Cytosine and Thymine
What are the 2 types of DNA bases?
Purines and pyrimidines
Which bases are purines?
Adenine and Guanine
Which bases are pyrimidines?
cytosine and thymine
What makes a base a purine?
They’re made up of 2 carbon (2C) rings
What makes a base a pyrimidine?
They’re made up of 1 carbon (1C) ring
Which part of the base attaches to the sugar in DNA?
the nitrogen
What type of bond joins the base and the sugar in a DNA nucleotide?
A B-N-glycosidic bond
What’s the full name of the nucleotide with an A base?
2’-deoxyadenosine 5’-triphosphate (dATP)
What’s the full name of the nucleotide with a G base?
2’-deoxyguanosine 5’-triphosphate (dGTP)
What’s the full name of the nucleotide with a C base?
2’-deoxycytidine 5’-triphosphate (dCTP)
What’s the full name of the nucleotide with a T base?
2’- deoxythymidine 5’-triphosphate (dTTP)
What happens to the phosphate group of a nucleotide when DNA is polymerising?
the alpha phosphate joins to the base of the other nucleotide and the B and Y phosphates are lost
What sort of bond joins 2 nucleotides together in DNA?
phosphodiester bond
Is DNA double or single stranded?
double
what shape does the double stranded DNA make?
A helix
Who discovered the double helix structure of DNA?
Watson, Crick and Franklin
Are the ends of DNA the same or different?
different
Does DNA have directionality?
yes
can DNA be read in 1 or 2 directions?
1
How is the directionality of the 2 strands in a DNA molecule described? what does this mean?
Antiparrallel organisation
This means they go in opposite directions- 1 going 5’ -> 3’ on one and 3’ –> 5’ on the other
Is DNA a regular helix?
no
What are the 2 grooves in DNA?
Major and Minor
What’s the advantage of having a major DNA groove?
enzymes can go in and read the DNA
What bonds hold the 2 strands of DNA together?
Hydrogen bonds
What’s the hydrogen bonding between the 2 strands of DNA called?
complementary base pairing
What bases pair?
A to T
C to G
How many hydrogen bonds between A and T?
2
How many hydrogen bonds between G and C?
3
Which is a stronger bond, that between A and T or that between G and C and why?
G and C has they have more hydrogen bonds
What do you call the amount of G + C nucleotides in an organism’s DNA?
GC content
What’s the GC content of human DNA?
40.3%- medium amount
can the DNA helix exist in different forms?
yes
What are the 3 forms of DNA helix?
A, B and Z
What’s the main difference between A and B helix DNA?
A DNA has a narrow major groove but B DNA has a wide major groove
Which is the best known DNA helix?
B
In what conditions does A-DNA form?
When there;s not enough water (dehydrated conditions)
What’s the structure of Z-DNA?
It’s wound into a left handed helix
Where’s Z-DNA found?
Found with B-DNA naturally in regions of human and other organism’s DNA
Is Z-DNA biologically active?
yes
What’s a Hydrogen bond?
A weak electrostatic attraction between an electronegative atom and a hydrogen atom attached to a second electronegative atom
What’s the 3’-OH terminus?
The end of a polynucleotide that terminates with a hydroxyl group attached to the 3’-carbon of the sugar
What’s the 5’- P terminus?
The end of a polynucleotide that terminates with a mono-, di- or tri- phosphate attached to the 5’- carbon of the sugar
What are the 2 roles of the genes in a cell?
- units of biological information
- genes are units of inheritance
what’s intergenic DNA?
part of DNA which aren’t genes
what’s the purpose of intergenic DNA?
mostly unknown, however believed to be involved in DNA replication
How long is a gene?
varies greatly
what’s the unit of length of a DNA molecule?
Base Pair (number of base pairs) (BP)
How many BPs in 1 kilobase pair (kb)?
1000000 (million) BP
How many BPs in 1 megabase pair (mb)?
1000 BP (1 million)
How long are the shortest genes?
around 100 bps
How long are the longest genes?
around 2,400,00 bp
Do the longest genes contain 24000 times more information than the shortest?
no
What are genes split into (to make them discontinuous)?
introns and exons
what are exons?
parts of the gene which contain the information needed to make protein
what are introns?
information not needed to make a protein
How many introns does the average human gene have?
8
How many exons does the average human gene have?
9
What’s the mean intron length?
3365 bp
what’s the mean exon length?
145 bp
are genes directional?
yes
which direction are genes read in?
5’–> 3’
What’s the process of reading gene information called?
gene expression
by which process do genes become RNA?
transcription
By which process does RNA become a protein?
translation
what are RNA and proteins?
functional end products of gene expression (sometimes RNA is the end product)
do proteins play many roles in the cell?
yes
name 3 types of protein..
any 3 from:
- structural proteins
- motor proteins
- catalytic proteins (enzymes)
- transport proteins
- storage proteins
- protective proteins
- regulatory proteins
Give an example of a structural protein
either collagen or keratin
give an example of a motor protein
myosin or dynein
give an example of an enzyme
hexokinase or DNA polymerase
give an example of a transport protein
hemoglobin or serum albumin
give an example of a storage protein
ovalbumin or ferritin
give an example of a protective protein
immunoglobulins or thrombin
give an example of a regulatory protein
insulin or somatostatin or somatotrophin
what’s the structure of collagen?
long and thin
what does hemoglobin do?
transports O2 in RBCs
what does insulin do?
regulates sugar levels
do RNAs that do not translate play important roles in cells?
yes
what percent of RNA is coding RNA?
4%
what’s the coding RNA?
mRNA (messenger)
what percent of RNA is noncoding?
96%
give 3 types of noncoding RNA
any 3 from
- rRNA
- tRNA
- snRNA
- snoRNA
- scRNA
- miRNA
- siRNA
What’s rRNA?
ribosomal RNA
what’s snRNA?
small nuclear RNA
What’s tRNA?
transfer RNA
where are noncoding RNAs often found?
in intergenic regions
what does it mean if a gene is unique?
there’s only one copy of this gene
what’s a gene family?
a group of the same/similar genes
when do simple multi-gene families occur?
when genes are needed in large quantities
what’s a complex multi-gene family?
where the genes aren’t identical but have similar sequences- they code for similar but non- identical proteins
what’s a tandem array?
a set of identical or very similar genes that are arranged one after the other in a group
do human globin genes form complex or simple multigene families?
complex
how many complex multigene families do globin genes form?
2
what are the 2 complex multigene families and where are they found?
the alpha-globin are on chromosome 16
the beta-globin are on chromosome 11
when are hemoglobin genes expressed in humans?
at different stages in human development
By what process do genes in a multigene family arise?
gene duplication
what happens after gene duplication?
the sequences of the genes gradually change so the members of the gene family have slightly different sequences
the greater the time that has passed since the duplication….
… the more differences we see between the pair of genes
why do we see more differences between gene pairs after a longer time has passed?
more mutations to the base code have occured
what’s a molecular clock?
a measure of the rate at which the sequence of a gene changes
what can molecular clocks show?
when a pair of genes were formed by duplication, allowing estimation of how long ago genes duplicated and diverted from each other (can study evolution)
what do you call genes that have lost their function?
pseudogenes
can pseudogenes become functional genes again?
yes, by mutation
How many pseudogenes are there in the alpha-globin family?
3
how many pseudogenes are there in the beta-globin familiy?
1
how does DNA replicate?
by semi-conservative replication
what is semi-conservative replication?
replication whereby half of the old information is included in the new molecule and the other half is new (gives 2 exact copies)
what were the other 2 possibilities of a mechanism for replication?
conservative and dispersive
How might conservative replication work?
- the parent molecule separates in to 2 strands
- semi-conservative replication occurs
- when strands rejoin the 2 old join together and the 2 new join together
what’s dispersive replication?
where old and new DNA are mixed up in both strands
How might dispersive replication happen?
the parent molecule opens up into 2 strands
- a bit of new DNA is made
- the template switches repeatedly and old and new segments join
why was semi- conservative replication first thought impossible?
DNA is plectonemic- strands can’t be separated without unwinding
many DNA molecules are circular making unwinding more difficult
what would happen if you made a double stranded cut to unwind circular DNA?
it would tighten up the helix
trying to unwind the DNA from one end would cause DNA at such a fast speed that the energy produced would destroy the DNA
which was the most popular theory of replication in the 1950s?
dispersive (due to the issue of unwinding)
What was the purpose of the meselson-stahl experiment?
to dsitinguish between the 3 modes of DNA replication and work out which occurs in E.coli bacteria
What was the method of the Meselson-Stahl experiment?
- A culture of bacteria were grown in a medium containing heavy Nitrogen (N15, instead of the normal N14)
- The bacteria colony was transferred to a growth medium containing normal Nitrogen (N14)
- Samples from this colony were removed after 20 minutes (1 cell division) and 40 minutes (2 cell division)
- The colonies were centrifuged
What were the findings of the Meselson-Stahl experiment?
- When grown in the heavy Nitrogen and centrifuged all bacteria appeared in the same band (15N), bacteria grown in normal Nitrogen (14) medium showed a higher up band (lighter)
- After one replication in lighter the medium, the bacteria band appeared in between where the 14 and 15 were
- after 2 replications, there was a band where N14 was and a band where the N14/15 was
Which method of replication did the meselson- stahl’s findings support?
semi-conservative
what would meselson- stahl have seen if they had been conservative replication after 1 division?
2 bands- one for N14, one for N15
what would meselson and stahl have seen after one replication in dispersive replication?
what they did see- a band in between N14 and N15
what would meselson- stahl have seen after two divisions in dispersive replication?
one band- between N14 and N15 (same as first division)
which type of enzyme is reponsible for DNA strand separation/ unwinding?
DNA topoisomerases
What’s the mode of action of a Type I DNA topoisomerase?
It makes a nick in 1 strand and passes the other through that nick to allow unwinding by releasing tension
What’s the mode of action of a Type II DNA topoisomerase?
- it makes a double strand nick and one segment of DNA passes through the gap, unwinding this section, then the 2 cut strands are rejoined and the process repeats further down the strand
Which enzyme breaks the bonds between the base-pairs?
DNA helicase
what’s the replication fork?
the open part of the DNA where the new strands of DNA are being synthesised
What’s the process of DNA synthesis called? (2 names)
template-dependent DNA synthesis
and
DNA-dependent DNA synthesis
What enzyme carries out DNA-dependent DNA synthesis?
DN-dependent DNA polymerase
What direction is DNA synthesis always in?
5’–>3’
what’s required to initiate the synthesis of a new DNA strand?
a primer
why is a primer needed to initiate the synthesis of a new DNA strand?
unknown
as well as synthesising DNA, what other function does DNA polymerase serve?
It can degrade DNA- exonuclease activity
How many types of exonuclease activity are there?
2
what are the names of the 2 types of exonuclease activity?
3’–>5’
and
5’–>3’
What happens during 3’–>5’ exonuclease activity?
the polymerase can remove nucleotides it has just just inserted
this is called proofreading, allowing errors to be corrected
What happens during 5’–>3’ exonuclease activity?
the polymerase can remove DNA already attached to the template
what are the 2 bacterial DNA polymerases?
I and III
what are the 2 eukaryotic DNA polymerases?
alpha (a) and delta
what’s the function of DNA polymerase I?
DNA repair and replication
What’s the function of DNA polymerase III?
main replicating enzyme- in bacteria
what’s the function of DNA polymerase alpha?
priming during replication
what’s the function of DNA polymerase delta?
main replicative enzyme- in eukaryotes
does DNA polymerase I have 3’–>5’ activity?
yes
does DNA polymerase I have 5’–>3’ activity?
yes
does DNA polymerase III have 3’–>5’ activity?
yes
does DNA polymerase III have 5’–>3’ activity?
no
does DNA polymerase alpha have 3’–>5’ activity?
no
does DNA polymerase alpha have 5’–>3’ activity?
no
does DNA polymerase delta have 3’–>5’ activity?
yes
does DNA polymerase delta have 5’–>3- activity?
no
what must first happen at the replication fork?
the separated single strands must be protected
why must the the separated single strands be protected? (2)
they may just reattach to each other
or
be attacked by nucleases
what protects the separated single strands at the replication fork?
single-strand binding proteins (SSBs)
What’s the leading strand?
The strand which can be synthesised by continuous DNA synthesis
Why can the leading strand be copied by continuous DNA synthesis?
It goes in the 5’–>3’ direction (with the parent strand being read 3’–>5’)
What’s the primer for bacteria synthesis made of?
RNA
In bacteria, what makes the primer?
Primase enzyme
how long is the primer in prokaryotes?
4-15 nucleotides in length
What synthesises the new DNA strand after the primer has been made in prokaryotes?
DNA polymerase III
what happens after the primer attaches to the template stand in eukaryotic DNA?
the primer is extended by DNA polymerase alpha, which adds about 20 nucleotides
then DNA polymerase delta makes the rest of the new strand
How is DNA on the lagging strand synthesised?- generally
It’s made in segments called Okazaki fragments
How are Okazaki fragments joined in bacteria?
DNA polymerase III stops when it reaches the RNA primer
DNA polymerase I continues synthesis
DNA ligase links the two DNA fragments
How are Okazaki fragments joined in eukaryotes?
DNA polymerase delta and helicase push aside the primer
FEN1 cuts the DNA at the branch point
there’s still a missing phosphodiester bond between the okazaki fragments which is joined by DNA ligase
What does the enzyme telomerase do in eukaryotes?
prevents the ends of chromosomes from getting shorter
How does the enzyme telomerase prevent the ends of chromosomes from getting shorter?
it extends the parent DNA by adding the sequence TTAGGG several times so the final okazaki fragment can be primed
What’s a genome?
the complete set of DNA molecules possessed by an organism
where does the replication of an E.coli genome begin?
the origin of replication (always the same position of the genome)
In E.coli, how is the direction of replication described?
bidirectional (goes in 2 directions)
describe the origin of replciation
- spans approx. 245 bp of DNA
- contains 2 short repeat motifs (one is 9 nucleotides long, the other is 13)
what are origins of replication like in human DNA?
there are many on each chromosomal DNA molecule
usually around 300kb apart from eachother
where do DNaA proteins bind?
close to the origin of replication
what happens to the DNA near the DNaA proteins?
it becomes wound around the barrel of these proteins
What does the winding of DNA around the DNaA proteins do?
causes torsional stress causing the base pairs at the origin of replication to break
what type of DNA is typically at the origin of replication? why?
A-T rich sequence
because the bonds are weaker (2 H bonds rather than 3)
How is the prepriming complex formed and opened into replication forks?
by attachment of DNaB proteins to the origin on either end. This starts breaking base-pair bonds, opening up the DNA further, moving the replication forks further away from the origin of replication
What is synthesised as the double helix unzips?
new polynucleotides
what is DNaB?
a helicase enzyme
How is the primosome formed?
by the attachment of 2 primase enzymes
What do primase enzymes do at the primosome?
make the RNA primers that initiate replication
what’s needed before DNA Polymerase III can begin to make DNA?
a special type of RNA polymerase that synthesizes the primers
describe events at the replication fork in E.coli?
- DNA topoisomerase separates the strands
- DNA helicase breaks base pairs
- single strand binding proteins (SSBs) protect the bare single strands
- primase makes primers on the lagging strand
- DNA polymerase III synthesizes DNA
- DNA polymerase I and DNA ligase joins up the okazaki fragment
- the gamma complex attaches and detaches polymerase III from the lagging strand
what’s single binding proteins (SSBs) purpose?(2)
prevent attacks by nucleases- if the body treats the exposed single strand as viral genetic material
prevents base pairs from rejoining
describe events at the replication fork in humans?
- DNA topoisomerases separates strands
- helicase breaks base pairs
- SSBs protect the single bare strands
- primase/DNA polymerase alpha make primers on the lagging strand
- DNA polymerase delta synthesizes DNA
- FEN1 and DNA ligase join up the okazaki fragments
- the proliferating cell nuclear antigen (PCNA) holds DNA polymerase delta tightly onto the DNA
What is required due to the bidirectionality of replication in E.coli?
a point to stop (termination point) at (in case the replication in either direction is going at different speeds)
How many terminator sequences are there in the E.coli genome?
6
what binds to each of the terminator sequences in E.coli?
Tus protein
are the terminator sequences in E.coli directional? why?
- yes
- because they affect the direction at which the Tus protein binds, one side has protein oriented one way the other the opposite way
What do Tus proteins allow and disallow?
replication fork can pass in one direction but not the other
what’s chromatin?
DNA extracted from the nucleus
a DNA-protein complex
what structure is shown on the electron microscope image of chromatin?
beads-on-a-string structure (proteins are beads on DNA string)
How are the proteins distributed along DNA?
At regular intervals
what are the proteins in chromatin called?
histones
what do histones contain in high amounts?
basic amino acid content
what’s a nucleosome?
the ‘beads’
an octamer of DNA wrapped around 8 histones
Which histones are found in a nucleosome?
2x H2A
2x H2B
2x H3
2x H4
How long is linker DNA between nucleosomes?
50-70 bp
What’s Histone H1?
a linker histone
what does Histone H1 do?
attaches to the outside of the nucleosome
what is a chromatosome made up of?
nucleosome + DNA + linker histone
Is DNA static?
no (during replication it leaves the nucleosome package and remains in it when not in use)
How much does the beads on a string structure reduce the length of DNA?
by 1/6
What’s the next level of packaging DNA?
30nm chromatin fibre
How much does the 30nm chromatin fibre reduce DNA length by?
another 1/7
are higher levels of packaging DNA after 30nm chromatin fibre understood?
no
what’s the highest level of DNA packaging?
the metaphase chromosome
where is the metaphase chromosome found?
only in dividing cells (when chromosomes condense)
How is euchromatin seen under the microscope?
light areas
what does euchromatin contain?
active genes
what level of packaging is euchromatin at?
probably 30nm fibre
How is heterochromatin seen under the microscope?
dark areas
what does heterochromatin contain?
inactive genes
Is heterochromatin or euchromatin more densely packed?
heterochromatin
what’s the difference between constitutive and facultative heterochromatin?
constitutive contains DNA that is tightly packed in all cells
facultative contains DNA that is tightly packed in some cells
what’s the nuclear matrix?
a protein network which gives round structure to nucleus
what is euchromatin attached to and why?
the nuclear matrix to prevent the DNA from getting tangled up
what’s the structure of a metaphase chromosome?
- centromere golds the daughter chromosomes together
telomeres protect the ends
what do the centromeres contain?
special histones- CENP-A instead of H3
what do telomeres protect the ends of chromosomes from?
exonuclease attack
and
being mistaken for chromosome breaks and joining together
what are chromatids?
the arms of chromosomes
what forms telomeres?
telomerase
what’s the karyogram?
a complete set of stained chromosomes
what does each chromosome show when stained?
a specific pattern of staining to that chromosome
what are the dark bands seen on chromosomes on the karyogram?
constitutive heterochromatin (where DNA/chromosomes are packaged more tightly)
define interphase
the period between cell divisions
what’s a nuclease protection experiment?
a technique that uses nuclease digestion to determine the positions of proteins on DNA or RNA molecules
What’s the UTR?
UnTranslated Region
what’s a spacer?
an area of non-coding DNA between genes (different to introns which are non-coding within genes)
what’s a cistron?
a section of DNA or RNA molecule that codes for a specific polypeptide in protein synthesis (a gene)
what 3 factors are required for the initiation of DNA replication in E.Coli?
Primase
DNA Helicase
DNaA
what’s unusual about telomerase?
it contains an RNA component
