7.1 DNA structure and replication Flashcards
structure of a nucleosome
DNA complexed with 8 histone proteins
how nucleosomes help supercoil DNA (DNA –> chromosome pipeline) 5
- nucleosomes (DNA wound around 8 histone proteins) are linked by additional histone protein to form string of CHROMATOSOMES
- string of chromatosomes coil to form a SOLENOID structure
- solenoid structure is condensed to form a 30 NM FIBRE
- fibres form loops, compressed and folded around protein scaffold to form CHROMATIN
- chromatin supercoils during cell dev to form CHROMOSOMES
Where does action of helicase occur
at specific regions (origins of replication), creating a REPLICATION FORK of 2 strands running in antiparallel directions
(helicase: unwinds and seps DNA by breaking H bonds betw base pairs)
what does DNA gyrase do
- reduces torsional strain created by unwinding of DNA by helicase
- relaxes pos supercoils (via neg supercoiling)
what do single stranded binding (SSB) proteins do
- SSB proteins bind to DNA strands after seperation, prevent them from re-annealing
- prevent single stranded DNA from being digested by nucleases
- will be dislodged from strand when a new complementary strand is synthesized by DNA polymerase iii
what does DNA primase do
- generates a short RNA primer on each of the template strands
- the RNA primer provides initiation point for DNA polymerase iii (can extend chain but not start)
how does DNA polymerase iii work
- free nucleotides align w complementary bases
- DNA pol iii attaches to 3’ END of primer
- covalently joins free nucleotides tgt in a 5’ –> 3’ DIRECTION
- moves in opp directions on 2 strands as they r antiparallel
DNA polymerase iii differences on leading and lagging strand
leading
- moves towards replication fork, synthesises continuously
lagging
- moves away from replication fork, synthesises in pieces (okazaki fragments)
what does DNA polymerase 1 do
- lagging strand is synthesised in a series of short fragments = has multiple RNA primers along its length
- DNA pol 1 removes the RNA primers from lagging strand and replaces them w DNA nucleotides
what does DNA ligase do
- joins the okazaki fragments tgt to form a continuous strand
- by covalently joining the sugar-phosphate backbones tgt w a phosphodiester bond
can DNA polymerase initiate replication? why
NO
- it can only add new nucleotides to an existing strand
- RNA primer must first be synthesised to provide an attachment point for DNA polymerase
DNA polymerase adds nucleotide to – end of the primer, extending the new chain in a —- direction
3’ end of primer
replication in a 5’ –> 3’ direction
free nucleotides exist as
deoxynucleoSIDE triphosphates (dNTPs) – have 3 phosphate grps
what cleaves the 2 additional phosphates from free nucleotides
DNA polymerase
uses energy released to form phosphodiester bond with 3’ end
how are okazaki fragments joined
on lagging strand
- fragments are preceded by a primer
- primers are replaced by DNA bases
- fragments joined tgt by combination of DNA pol 1 and DNA ligase
4 examples of non coding DNA
- regulators of gene expression
- introns
- telomeres
- genes for tRNA
non coding dna – regulators of gene expression what are they + 3 examples
DNA sequences that regulate gene expression
- promoters: sequences that occur just before genes, act as a binding point for RNA polymerase enzymes that catalyse transcription
- enhancers: DNA sequences that act as binding sites for protein – INCR rate of transcription
- silencers: DNA sequences that act as binding sites for proteins – DECR rate of transcription
what are introns
DNA base sequences found in eukaryotic genes – get removed at the end of transcriptions
(do not contribtue to the AA sequence transcribed)
what are telomeres
repetitive sequences that protect the ends of the chromosome
- ensure DNA is replicated correctly (every cell div DNA is lost from telomeres)
- protects against chromosomal deterioration during replication
what protects against chromosomal deterioration during replication
telomeres (repetitive sequences at ends of chromosome)
non coding region: genes for tRNA
code for RNA molc – do not get translated into proteins
- fold to form tRNA molc that play impt role in translation
how was X-ray diffraction carried out? (rosalind franklin and maurice wilkin)
- DNA purified, fibres stretched in thin glass tube (make strands parallel)
- DNA targeted by X ray beam – diffracted when contacted an atom
- scattering pattern of X ray recorded and used to investigate molecular structure
inferences from rosalind franklin and maurice wilkin’s investigation of DNA structure by X-ray diffraction 3
- composition
DNA is double stranded - orientation
nitrogenous bases are closely packed tgt inside, phospphate backbone outside - shape
DNA twists at regular intervals (34 angstrom) to form helix (2 strands = double)
what is DNA sequencing
process by which base order of a nucleotide sequence is elucidated
what nucleotides are used to stop DNA replication
dideoxynucelotides
DI DEOXYNUCLEO TIDES
How do dideoxynucleotides stop DNA replication
- they lack the 3’-hydroxyl grp needed to form a phosphodiester bond
- prevent further elongation, terminating replication
what is sanger method for
base sequencing
sanger method steps 4
- four PCR mixes, each w stocks of normal nucleotides and one dideoxynucleotide (ddA, ddT, ddC, ddG)
- each mix generates all possible terminating fragments for that base
- fragments seperated using gel electrophoresis
- base sequence determined by ordering fragments acc to length
sanger method: fragments can be detected by automated sorting machines if
a distinct radioactive or fluorescently labelled primer is included in each mix
if the sanger method is conducted on the coding strand (non template), the resulting sequence will be
identical to the template strand
what are tandem repeats used in
DNA profiling – indivs can be identified and compared by analysing respective DNA profiles
what are tandem repeats
- a sequence of 2 or more DNA base pairs – repeated in such a way that the repeats lie end-to-end on the chromosome
- within non-coding regions there is SATELLITE dna – long streches w repeating short tandem repeats (STR)
how are tandem repeats taken for comparison
repeats can be excised using restriction enzymes, then seperated with gel electrophoresis
why do tandem repeats generate unique DNA profiles
indivs have diff numbers of repeats at a given satellite DNA locus
experiment that proved DNA was genetic material
Hershey and Chase experiment
Hershey and Chase experiment steps 5
- viruses grown in 1 of 2 isotopic mediums to radioactively label a specific viral component : radioactive sulfur = proteins, radioactive phosphorus = DNA
- viruses infected bacterium then seperated via centrifugation
- larger bacteria formed solid pellet, smaller viruses remained in supernatant
- bacterial pallet found to be radioactive when infected by DNA virus but not protein
- proves DNA was genetic material bc it was transferred to the bacteria
nucleosome structure
- molecule of DNA wrapped around 8 histone proteins (octamer)
- negatively charged DNA associates w positively charged AA on surface of histone proteins
- histone proteins have N-terminal tails which extrude from nucleosome
- chromosomal condensation: tails from adjacent histone octamers link up and draw nucleosomes tgt
