Lecture 13 Flashcards
(38 cards)
1
Q
Requirements for replication
A
- the process must be very accurate
- the process must be very fast
2
Q
Meselson-Stahl experiment
A
- used isotopes 14N and 15N and the technique of equilibrium density centrifugation
- E. Coli grown with N15 as sole nitrogen source
- after many generation, all of the DNA had incorporated 15N.
- Removed the sample to only 14N and took samples.
- first replication with 14N proved replication wasn’t conservative but didn’t prove if it was semiconservative or disruptive
- second replication proved DNA replication is semiconservative
3
Q
origin of replication
A
where replication starts
4
Q
theta replication
A
- double stranded DNA begins to unwind at the replication origin, producing single stranded strands that serve as templates for replication
- unwinding generates a replication bubble
- replication on both template strands is simultaneous with unwinding
- usually a replication fork at the end of each bubble
- produces two circular DNA molecules
- BIDIRECTIONAL CIRCULAR REPLICATION
5
Q
Rolling circle replication
A
- replication is initiated by a break in one of the strands that creates a 3’ -OH group and a 5’-phosphate group
- new nucleotides are added to the 3’ end. the 5’ end of the broken strand is displaced from the template, rolling out like thread being pulled off a spool. The 3’ end grows around the circle.
- UNIDIRECTIONAL
6
Q
Linear eukaryotic replication
A
- replication originates at multiple origins
- at each replication origin, the DNA unwinds and produces a replication bubble, with the two replication forks spreading outward.
- eventually, the replication forks of adjacent replicons run into each other, and the replicons fuse to form long stretches of newly synthesized DNA.
- products are two linear DNA molecules
7
Q
Requirements of replication
A
- a template consisting of single-stranded DNA
- dNTPs complementary to template strand
- 3’ OH group on a primer
8
Q
dNTPS
A
- deoxyribonucleside triphosphates
- the raw materials from which new DNA is synthesized
- consists of a deoxyribose sugar and a base attached to three phosphate groups
9
Q
DNA synthesis
A
- nucleotides added to the 3’ OH group of the growing nucleotide strand
- The 3’ OH group of the last nucleotide attacks the 5’ phosphate group of the incoming dNTP
- two phosphate groups are cleaved from the incoming dNTP, and a phosphodiester bond is formed between the two nucleotides
10
Q
DNA polymerases
A
enzymes that synthesize DNA in the 5’ to 3’ direction
11
Q
leading strand
A
- exposed in the 3’ to 5’ direction
- undergoes continuous synthesis in the direction of replication
12
Q
lagging strand
A
- exposed in the 5’ to 3’ direction
- the newly made strand that undergoes discontinuous replication in the opposite direction as replication
13
Q
Okazaki fragments
A
- the short lengths of DNA produced by discontinuous replication of the lagging strands
14
Q
Initiation bacterial replication
A
- initiator proteins bind to oriC to cause a short section of DNA to unwind.
- a replication bubble with a replication fork on each side is created
- this unwinding allows other enzymes to attach to the strand
15
Q
DNA helicase
A
- breaks the hydrogen bonds between the bases of the two strands of DNA to unwind up
- cannot initiate unwinding. initiator protein must do that first
- binds to the lagging strand template and each replication fork and moves 5’ to 3’
16
Q
single-stranded-binding proteins
A
- attach to the single-stranded DNA in order to protect the chains and prevent the formation of secondary structures.
- keep the strands of the replication fork separated
17
Q
DNA gyrase
A
topoisomerase that moves ahead of the replication fork, making and resealing breaks in the double-helical DNA to release the torque that builds up as a result of unwinding at the replication fork.
- removes a twist in the DNA and reduces the supercoiling
18
Q
synthesis of primers
A
- all DNA polymerases require a nucleotide with a 3’ -OH group to which a new nucleotide can be added. This of this, DNA polymerases cannot initiate DNA on a bare template. They require a primer - an existing 3’ -OH to get started
19
Q
primase
A
- synthesizes short stretches of RNA nucleotides, or primers which provides a 3’ -OH group to which DNA polymerase can attach to get DNA replication started
- primase is an RNA polymerase so it does not require a 3’ -OH group to get started
20
Q
DNA polymerase III
A
- attaches to the primer and synthesizes strands by adding new nucleotides to the 3’ end of a growing DNA molecule until it reaches the next primer
- 5’ to 3’ activity to add nucleotides in the 5’ to 3’ direction
- 3’ to 5’ exonuclease activity to remove errors
21
Q
DNA polymerase I
A
- same activity as polymerase III but also has 5’ to 3’ exonuclease activity that allows it to remove the primers laid down by primes and replace them with DNA
22
Q
DNA ligase
A
joins Okazaki fragments by sealing nicks in the sugar-phosphate backbone of newly synthesized DNA
23
Q
Termination in bacterial replication
A
- terminated whenever two replication forks meet
- in others, specific termination sequences block further replication
24
Q
Differences in eukaryotic and bacterial DNA replication
A
- in eukaryotic cells, replication is coordinated with the cell cycle
- the important G1/S checkpoint holds the cell cycle in G1 until the DNA is ready to be replicated. After this checkpoint is passed, the cell enters S phase and the DNA is replicated
25
Licensing of DNA replication in Eukaryotic DNA replication
- origins are licensed or approved for replication. early in the cell cycle a replication licensing factor attaches to an origin
- replication machinery initiates replication at each licensed origin. as the replication forks move away from the origin, the licensing factor is removed, leaving the origin in an unlicensed state. replication cannot be reinitiated until the license is renewed
26
Eukaryotic DNA polymerases
- alpha
- delta
- epsilon
27
DNA polymerase alpha
- has primase activity; synthesizes an RNA primer followed by a short stretch of DNA
28
DNA polymerase delta
completes synthesis on the lagging strand
29
DNA polymerase epsilon
replicates the leading strand
30
Nucleosome Assembly
Occurs in three steps:
1. disruption of original nucleosomes on the parental DNA molecule ahead of the replication fork
2. the redistribution of preexisting histones on the new DNA molecules
3. the addition of newly synthesized histones to complete the formation of new nucleosomes
31
The end-replication problem
- in a circular DNA molecule, elongation around the circle eventually provides a 3' -OH group immediately in front of the primer
- After the primer has been removed, the replacement DNA nucleotides can be added to this 3' -OH group
32
Replication at the ends of chromosomes
- in linear chromosomes, elongation of DNA in adjacent replicons provides a 3' -OH group for replacement of each primer
- Primers at the ends of chromosomes cannot be replaced, because there is no adjacent 3' -OH to which DNA nucleotides can be attached
- When the primer at the end of a chromosome is removed there is no 3' -OH group to which DNA nucleotides can be attached, producing a gap at the end of a chromosome
suggests that chromosomes could become shorter with each round of replication
33
Telomeres
- presence of many copies of a short repeated sequence at the end of chromosomes
34
telomerase
- extend telomeres
- an enzyme with a protein and RNA component
- this sequence pairs with the overhanging 3' end of the DNA and provides a template for synthesis of additional DNA copies of the repeats
35
where is telomerase expressed?
- single celled organisms
- germ cells
- early embryonic cells
- certain proliferative somatic cells that must undergo continuous cell division
36
most somatic cells have
little or no telomerase activity, and chromosomes in these cell progressive shorten with each cell division
37
when the telomeres have shortened beyond critical length
the chromosome becomes unstable, has a tendency to undergo rearrangements and is degraded. cell death may occur due to these events.
38
Werner syndrome
- autosomal recessive disease in which premature agent begins in adolescence or early adulthood
- the causative gene has been mapped and normally encodes an enzyme necessary for the efficient replication of telomeres
