WEEK 8 (DNA Replication) Flashcards
Except in some viruses, DNA serves as the ____________________ in all living organisms on Earth
genetic material
According to the Watson-Crick model, DNA exists in the form of what?
a right-handed double helix
Describe the DNA structure
The strands of the double helix are antiparallel and are held together by hydrogen bonding between complementary nitrogenous bases
What does the structure of DNA provide the means of?
storing and expressing genetic information
Describe the study that became the first evidence that DNA could serve as the genetic material
Fred Griffith was studying STREPTOCOCCUS PNEUMONIAE (a bacterium that causes pneumonia). The experiment involved an injection into mice of living IIR (avirulent) [not pathogenic] cells combine with heat-killed IIIS (virulent) cells. Griffith showed that heat-killed, infectious bacteria can transform harmless, living bacteria into pathogenic ones.
Later, researchers prepared an extract from the disease-causing S strain of pneumococci and showed that the “transforming principle” that would permanently change the harmless R-strain pneumococci into the pathogenic S strain is DNA
What does the Watson-Crick model predict?
It predicts that genetic continuity between parental and progeny cells is maintained by semiconservative replication of DNA
Describe the Semiconservative replication of DNA
Semiconservative replication uses each strand of the parent double helix as a template, and each newly replicated double helix includes one “old” and one “new” strand of DNA
What does the complementarity of DNA strands allow?
It allows each strand to save as a template for synthesis of the other
What are the three possible modes by which replication could be accomplished?
- Semi-conservative
- Conservative
- Dispersive
In 1958, Matthew Meselson and Franklin Stahl published the results of what?
an experiment providing strong evidence that semiconservative replication is the mode used by bacterial cells to produce new DNA molecules
Describe what happened in the Meselson-Stahl experiment
E-coli DNA was uniformly labelled with 15N nitrogenous bases by being grown in a 15N-labeled medium. This medium was then added to a 14N-labeled medium and the two cells were left to replicate. With the prediction of conservative replication, two distinct bands was expected; however, after the first generation only one intermediate band occurred between 14N and 15N. After two cell divisions, DNA samples showed two density bands- one intermediate and one lighter band corresponding to the 14N position in the gradient. After the third generation, the proportion of the lighter band increased.
This was evidence of semiconservative replication
Describe the Taylor-Woods-Hughes experiment
The Taylor-Woods-Hughes experiment demonstrates the semiconservative mode of replication of DNA in root tips of Vicia faba
1) An unlabelled chromosome proceeds through the cell cycle in the presence of 3H-THYMIDINE. As it enters mitosis, both sister chromatids of the chromosome are labelled by AUTORADIOGRAPHY.
2) After a second round of replication, this time in the absence of 3H-THYMIDINE, only one chromatid of each chromosome is expected to be surrounded by grains. (except where a reciprocal exchange has occurred between sister chromatids)
the expectation was upheld and was evidence of semiconservative replication
What are the two experiments that lead to the general acceptance of the Semiconservative mode of replication
Meselson-Stahl experiment & Taylor-Woods-Hughes experiment
Where does DNA replication begin?
at the origin of replication
Define Replicon
A replicon is the length of DNA that is replicated following one initiation event at a single origin
Why is the rate of fork movement slower in eukaryotes than in prokaryotes?
It may be due to the difficulties in replicating DNA through the more complex chromatin structure of eukaryotic chromosomes
DNA molecules in the process of being replicated contain Y-shaped junctions called ______________________
replication forks
How many replication forks are formed at each replication origin?
2
Describe what happens at the origin of replication
1) DNA molecules in the process of being replicated contain Y-SHAPED JUNCTIONS called REPLICATION FORKS. Two replication forks are formed at each replication origin.
2) At each fork, a replication machine moves along the DNA, opening up the two strands of the double helix and using each strand as a template to make a new daughter strand
3) The two forks move away from the origin in OPPOSITE DIRECTIONS, unzipping the DNA double helix and replicating the DNA as they go
Why is DNA replication in bacterial and eukaryotic chromosomes termed ‘bidirectional’?
Since the two forks move away from the origin in opposite directions, unzipping the DNA double helix and replicating the DNA as they go
What are the 7 key issues that must be resolved during DNA replication?
- unwinding of the helix
- reducing increased coiling generated during unwinding
- synthesis of a primer for initiation
- discontinuous synthesis of the second strand
- removal of the RNA primers
- joining of the gap-filling DNA to the adjacent strand
- proofreading
Why can only one strand of DNA serve as a template for continuous DNA synthesis?
The two strands of a double helix are ANTIPARALLEL to each other since one runs in the 5’ to 3’ direction and the other has the opposite 3’ to 5’ polarity. Since DNA polymerase III synthesises DNA in only the 5’ to 3’ direction, synthesis along an advancing replication fork occurs in one direction on one strand and in the opposite direction on the other. As a result, as the strands unwind and the replication fork progresses down the helix, only one strand can serve as a template for continuous DNA synthesis.
What is the name of the newly synthesised DNA?
Leading strand
What results in the discontinuous DNA synthesis of the lagging strand?
As the fork progresses, many points of initiation are necessary on the opposite DNA template, resulting in discontinuous DNA synthesis of the lagging strand
Why is opposite polarity of synthesis along the two strands of DNA necessary?
Because the two strands run parallel to one another and because DNA polymerase III synthesises in only one direction (5’ to 3’)
What does the discontinuous synthesis on the lagging strand result in?
The production of Okazaki fragments
What are RNA primers used for?
To initiate synthesis on both strands since it brings new nucleotides and checks that these nucleotides are complementary to the strand
Describe the stages of DNA replication occurring at a single replication fork
1) At the advancing fork, a HELICASE in unwinding the double helix and once unwound, SINGLE-STRANDED BINDING PROTEINS associate with the strands preventing the reformation of the helix
2) In advance of the replication fork, DNA GYRASE functions to diminish the tension created as the helix supercoils
3) Each of the core enzymes of DNA POLYMERASE III holoenzyme is bound to one of the template strands by a sliding DNA clamp
4) Continuous synthesis occurs on the leading strand, while the lagging strands must loop around in order for simultaneous synthesis to occur on both strands
5) Essential to replication on the lagging strand is DNA POLYMERASE I and DNA LIGASE which together replace the RNA primers with DNA and join the OKAZAKI FRAGMENTS respectively
To elongate a polynucleotide chain, what does DNA polymerase III require?
A primer with a free 3’-OH group
What are the key components of DNA in eukaryotic cells?
- There is more DNA than prokaryotic cells
- The chromosomes are linear
- The DNA is complexed with proteins
- Eukaryotic chromosomes contain multiple origins of replication to allow the genome to be replicated in a few hours
What are the differences between Prokaryotic and Eukaryotic replication?
PROKARYOTES
- ORIGIN OF REPLICATION: single
- RATE OF REPLICATION: 1000 nucleotides
- DNA POLYMERASE TYPES: 5
- DNA PACKAGING: supercoiling
- TELOMERASE: not present
EUKARYOTES
- ORIGIN OF REPLICATION: multiple
- RATE OF REPLICATION: 50 to 100 nucleotides
- DNA POLYMERASE TYPES: 14
- DNA PACKAGING: wound around histones
- TELOMERASE: present
How do eukaryotic cells facilitate the rapid synthesis of large quantities of DNA?
eukaryotic chromosomes contain multiple replication origins
What are the roles of eukaryotic replication origins?
eukaryotic replication origins not only act as sites of replication initiation, but also control the timing of DNA replication
What are regulatory functions carried out by?
regulatory functions are carried out by a complex of more than 20 proteins called PREREPLICATION COMPLEX (pre-RC), which assembles at replication origins
What happens in the early G1 phase of the cell cycle?
In the early G1 phase of the cell cycle, replication origins are recognised by a six protein complex known as an ORIGIN RECOGNITION COMPLEX (ORC) which tags the origin as a site of initiation of replication
The human genome contains genes that encode how many different DNA polymerases?
at least 14 different polymerases
only three of which are involved in the majority of nuclear genome DNA replication
What are polymerases alpha, delta and epsilon?
The major forms of the enzyme involved in initiation and elongation during eukaryotic nuclear DNA synthesis
Two of the four subunits of the ________________ synthesise RNA primers on both the leading and lagging strands
Polymerase alpha enzyme
What are the functions of DNA alpha polymerase?
RNA/DNA primers & initiation of DNA synthesis
What are the functions of DNA delta polymerase?
- lagging strand synthesis
- repair
- recombination
- proofreading
What are the functions of DNA epsilon polymerase?
- leading strand synthesis
- repair
- recombination
- proofreading
What is the function of DNA gamma polymerase?
Mitochondrial DNA replication & repair
What is the function of DNA beta polymerase?
Base-excision DNA repair
Which DNA polymerases are involved in Translesion DNA synthesis?
- eta
- zeta
- kappa
- nu
- iota
Which DNA polymerases are involved in DNA repair?
- theta
- lambda
- mu
- nu
- Rev 1
When does DNA replication occur?
During the S phase of the cell cycle
What does ‘semi-conservative’ mean?
It produces two copies that each contain one of the original strands and one new strand
What is the difference in origin of replication for bacteria and eukaryotes?
There is only a single origin of replication in bacteria and there are multiple origins of replication in eukaryotes
How does DNA replication take place?
In a 5’ to 3’ direction with a leading strand and a lagging strand (which is discontinuous) and the use of an RNA primer
The rate of replication in eukaryotic cells is __________ than the rate in prokaryotic cells
lower
Why are Primers used to initiate synthesis on RNA molecules?
only RNA has a free OH group
What does 5’ to 3’ mean?
The 5’ and 3’ designations refer to the number of carbon atom in a deoxyribose sugar molecule to which a phosphate group bonds.
Which enzyme reduces coiling?
Topoisomerase
How do prokaryotes package their DNA?
Supercoiling
What are the differences between prokaryotes & eukaryotes?
PROKARYOTES
- single origin of replication
- 5 types of DNA polymerase
- higher rate of replication
- packs DNA via supercoiling
-Telomerase not present
EUKARYOTES
- multiple origins of replication
- 14 types of DNA replication
- lower rate of replication
- packs DNA by wounding around histones
- Telomerase present
Why don’t prokaryotic cells contain Telomerase?
Since Prokaryotic DNA is circular therefore it has no ends
What is the difference between transcription of DNA in the Nucleus and the Mitochondria?
In the Nucleus only one strand is used as a template strand; In Mitochondria both strands are used in transcription
ATP is a __________________
Nucleotide
Describe Phosphodiester linkage
3’-hydroxyl group of one nucleotide unit is joined to the 5’-phosphate group of the next nucleotide
According to Chargaff’s rule, in a DNA molecule __________________
The amount of adenine and thymine is equal to the amount of guanine and cytosine
Which DNA helix has alternating GC sequences?
Z-DNA Helix
