Genetics - DNA replication Flashcards
what important tasks do cells need to complete before they divide?
grow
copy its genetic material (DNA)
physically split into two daughter cells
what two major phases is the cell cycle divided into?
interphase and mitosis (M) phase
what happens during interphase?
G1, S, G2
the cell grows and makes a copy of its DNA
During the mitotic phase, what happens?
the cell separates its DNA into two sets and divides its cytoplasm, forming two new cells
at each cell division, what must a cell do?
it must copy its genome
it is essential that this process occurs accurately
Semi-conservative replication?
DNA acts as a template for its own replication
what type of replication does DNA replicate by?
semi-conservative replication
what does DNA helicase do?
unwinds the DNA
What does DNA polymerase do?
synthesises DNA 5’-3’ direction
what does DNA topoisomerase do?
relieves the tension in DNA
What does DNA primase do?
synthesises RNA primers
what do ribonucleases do?
degrades RNA primers
what does DNA ligase do?
joins DNA fragments
what does telomerase do?
replicates the ends of the chromosome
DNA unwinding?
DNA helicase
SSB
DNA topoisomerase
DNA helicase function?
unwinds the DNA
uses ATP to propel itself along the DNA
SSB? Function?
SSB - single-stranded DNA binding protein
Binds and keep the strands apart
DNA topoisomerase function?
relieves the tension
DNA synthesis process?
DNA polymerase requires a template
DNA polymerase requires a primer
A new strand of DNA is always synthesised in a 5’ to 3’ direction
it elongates from a free 3’OH
Synthesis begins at an origin replication
DNA primase?
synthesises short RNA primer
primer?
short segment of RNA complementary to the template with a 3’OH
3’OH?
3 prime hydroxyl group
DNA replication progresses in the –>
5’-3’ so replication on the leading strand is continuous
As DNA replication cannot progress in the opposite direction…
replication on the lagging strand is discontinuous
Okazaki fragments?
the short DNA sequences (100-1000 nucleotides) synthesised on the lagging strand
DNA helicase results in the formation of a…
replication fork
exonuclease?
removes all of the RNA primers –> another DNA polymerase molecule fills these gaps with DNA nucleotides
How does the DNA polymerase remain attached to the DNA template?
by interaction with a protein called sliding clamp
when does a new sliding clamp come in?
a new clamp has to be loaded on the lagging strand as each Okazaki fragment is synthesised
What happens with the lagging strand?
DNA primase attaches RNA to template
DNA polymerase III adds nucleotides until it reaches the previous primer
RNase H digests the RNA primer, leaving a gap
DNA polymerase I fills in the gaps
DNA ligase then joins the fragments together
telomerase?
elongates parental strand
end problem
what are telomeres?
repetitive regions at the very ends of chromosomes are called telomeres
what do telomeres act as?
act as caps that protect the internal regions of the chromosomes, and they’re worn down a small amount in each round of DNA replication
Depurination?
no DNA breaks
removal of purines
removal of guanine or adenine base to leave sugar-phosphate group
Deamination?
no DNA breaks
results in a C to U transition
e.g. conversion of cytosine to uracil
What is a gain of function mutation?
A DNA sequence change that leads to increased or alternative activity
What is a loss of function mutation?
A DNA sequence change that leads to decreased activity
silent mutation?
when a nucleotide substitution results in a different codon that still encodes the same amino acid - therefore the protein is unaffected in function and the phenotype of the organism is not significantly altered
Missense mutation?
when a nucleotide substitution results in a codon that encodes a different amino acid - therefore the primary protein sequence is altered which may be conservative or radical
what is a conservative substitution?
similar amino acid R group size and charge
similar protein shape and function
e.g. Gly –> Ala
radical substitution?
new amino acid R group different in charge or size
protein may have altered secondary or tertiary structure affecting function
consequences of missense mutation?
conservative substitution
radical substitution
disrupting functional or structurally important residues
Nonsense mutations?
when a nucleotide substitution results in a stop codon that stops translation, therefore the protein truncated and may not function properly or even at all
what is protein truncation?
shortening due to change of sequence resulting in stop codon
NMD?
nonsense mediated decay
a process during translation that detects transcripts with premature stop codons and degrades them
3 mechanisms for DNA repair
Excision
Repair
Joining
excision?
recognition and removal of damage
exonuclease
repair?
re-synthesis of missing DNA
DNA polymerase
Joining?
sealing the nick
DNA ligase
Mismatch repair?
DNA replication makes a mistake 1 in 10(7) nucleotides copied - mismatch repair reduces this to 1 in 10(9)
What do RNA primers provide when we are creating the lagging strand?
the 3’ OH groups at regular intervals along the lagging strand template
when does the lagging strand stop?
it stops short of the end of the lagging strand template
why do chromosomes get progressively shorter during each replication cycle?
because there is an inability to replicate the ends as there is
no 3’ OH group available to prime DNA synthesis
what solves the end replication problem?
solved by the enzyme telomerase
telomere?
G-rich series of repeats
where is a telomere found?
in the ends of chromosomes
what does telomerase do?
recognises the tip of an existing repeat sequence, using an RNA template within the enzyme
telomerase elongates the parental strand in the 5’ to 3’ direction and adds additional repeats as it moves down the parental strand
how is the lagging strand completed?
by DNA polymerase alpha, carries a DNA primase as one of its subunits - allows the ends to be completely copied in the new DNA
What is the problem in the lagging strand?
Okazaki fragments cannot cover the end of the chromosome
Why can the other primers throughout the lagging strand be replaced, apart from the end one?
there is no way to get the fragment started because the primer would fall beyond the chromosome end
What happens in each round of replication because of the problem in the telomeres?
part of the DNA at the end of a eukaryotic chromosome goes uncopied in each round of replication
part of the DNA at the end of a eukaryotic chromosome goes uncopied in each round of replication - what does this result in?
leaves a single-stranded overhang.
part of the DNA at the end of a eukaryotic chromosome goes uncopied in each round of replication - what happens over multiple rounds of cell division?
the chromosome will get shorter as this process repeats
what is telomerase?
an enzyme which is an RNA-dependent DNA polymerase, meaning an enzyme that can make DNA using RNA as a template
what is the solution for the problem caused in telomeres?
the enzyme binds to a special RNA molecule that contains the sequence complementary to the telomeric repeat
telomere solution, the enzyme binds to a special RNA molecule that contains the sequence complementary to the telomeric repeat - what happens after this?
telomerase recognises the tip of an existing repeat sequence and uses the RNA template within the enzyme to add additional repeats to the telomere DNA
Telomere - solution, telomerase recognises the tip of an existing repeat sequence and uses the RNA template within the enzyme to add additional repeats to the telomere DNA - what happens after this?
when the overhang is long enough, a matching strand can be made by DNA polymerase alpha, which has its own primase subunit - so DOESN’T NEED A PRIMER
how does the active site help with ‘proof-reading’ and ‘fidelity’?
active site geometry only accommodates A-T & G-C base pairs
How is the ‘proof-reading’ carried out?
3’ to 5’ exonuclease activity
what can cause damage to DNA?
can be damaged as a result of UV light, ionising radiation exposure, toxic chemical agents, reactive oxygen species
What can DNA damage result in?
results in mutations in genes that can lead to altered coding for proteins resulting in loss or gain of functions
how many errors does a typical mammalian cell accumulate in its DNA every 24 hours?
many thousands of errors
What type of damage arises from UV light exposure?
Thymidine Dimers
What are the types of damages to the DNA?
Double stranded breaks, thymidine dimers, depurination, deamination
What kind of damage are we talking about when we say ‘thymidine dimers’?
two adjacent thymine bases become covalently attached to each other –> leads to stalling of the replication machinery
Failure to repair thymidine dimer leads to?
this is the problem in xeroderma pigmentosum
what causes double stranded breaks?
some environmental factors such as high energy radiation
what happens during double stranded breaks?
chromosomes are literally split in two
In double stranded breaks, what depends on where the break is and how extensive the damage is?
depending on these two factors, large numbers and the information they encode can be lost
