Chapter 11 Flashcards
What is DNA replication?
The process in which original DNA strands are used as templates for the synthesis of new DNA strands.
DNA replication relies on what?
The complementarity of DNA strands according to the AT/GC rule.
What are template strands?
In DNA replication, the DNA strand that is used as a template.
What’s another name for template strand?
Parental strand
After the double helix have separated, what happens?
Individual nucleotides have access to the template strands.
Hydrogen bonding between individual nucleotides and the template strands must obey what?
The AT/GC rule
How is the replication process complete?
A covalent bond is formed between the phosphate of one nucleotide and the sugar of the previous nucleotide.
The two newly made strands are referred to as what?
Daughter strands.
DNA replication results in both copies what?
Retaining the same information- the same base sequence- as the original molecule.
What features of DNA structure enable it to be replicated?
Its double-stranded structure
The base pairing between A and T and between G and C
What are the three different mechanisms to explain the net result of DNA replication?
Conservative Model
Semiconservative Model
Dispersive Model
What is the conservative model?
An incorrect model in which both strands of parental DNA remain together following DNA replication.
In the conservative model what is the result?
The original arrangment of parental DNA remain together while the two newly made daugther strands remain together.
What is the semiconservative model?
The correct model for DNA replication in which the newly made double-stranded DNA contains one parental strand and one daugther strand.
What is the result of the semiconservative model?
The newly made double-stranded DNA contains one parental strand and one daugther strand.
What is the dispersive model?
An incorrect model for DNA replication in which segments of parental DNA and newly made DNA are interspersed in both strands following the replication proccess.
What did Matthew Meselson and Franklin Stahl do as an experiment? When?
Devised a method to experimentally distinguish newly made daughter strands from the original parental strands in 1958
What was Meselson and Stahl’s technique?
Labeling DNA with a heavy isotope of nitrogen.
Why did Meselson and Stahl use nitrogen?
It is found in the bases of DNA in a heavy (15N) or light (14N) form.
What did Meselson and Stahl do prior to their experiment?
They grew E coli cells in the presence of 15N for many generations. So all the DNA was heavy-labeled.
At the start of Meselson’s and Stahl’s experiment what did they do?
They switched the bacteria to a medium that contained only 14N and then collected sample of cells after various time points.
What happened during Meselson’s and Stahl’s experiement?
Under the growth conditions, the cells replicated their DNA and divided into daugther cells every 30 minutes. After each cell doubling, the new daugther cells were viewed. The new daughter cells were labeled with light nitrogen, while the original strands remained heavy.
How did Meselson and Stahl analyze the density of the DNA?
By centrifugation, using a ceisum chloride gradient. If both strands were light they would be at the top. If both strands were heavy they would be at the bottom, if there was one of each they would be in the middle.
After two rounds of DNA replication, what did Meselson and Stahl find?
A mixture of light DNA and Half-heavy DNA which was consistent with the semiconservative model.
The complementarity of DNA strands is based on
a. the chemical properties of a phosphodiester linkage
b. the binding of proteins to the DNA
c. the AT/GC rule.
d. none of the above.
c. the AT/GC rule.
To make a new DNA strand, which of the following is necessary?
a. a template strand
b. nucleotides
c. heavy nitrogen
d. both a and b.
d. both a and b.
The model that correctly describes the process of DNA replication is
a. the conservative model
b. the semiconservative model
c. the dispersive model.
d. all of the above.
b. the semiconservative model
Bacterial chromosomes have a single what?
Origin of replication.
The synthesis of new daughter strands is initiated how?
Within the origin and proceeds in both directions.
What is bidirectionally?
The phenomenon in which two replication forks move in opposite directions, outward from the origin.
What are replication forks?
The regions in which two DNA strands have separated and new strands are being synthesized.
Eventually the replication forks do what?
Meed each other on the opposite side of the bacterial chromosome to complete the replication process.
What is the name of the origin of replication in E coli?
oriC (origin of Chromosomal Replication)
What are the three types of DNA sequences found in oriC?
An AT-rich region
DnaA box sequence
GATC methylation sites.
The AT-rich region is composed of what?
Three similar sequences that are 13bp long.
What are the functions of the AT-rich region and DnaA boxes?
The DnaA boxes are recognized by DnaA protein, which binds to them and causes the DNA to unwind at the At-rich region.
DNA replication is initated by what in bacterial chromosomes?
DnaA protein
What are DnaA proteins?
Proteins that binds to the DnaA box sequence at the origin of replication in bacteria and initiates DNA replication.
What are DnaA box sequences?
Serves as a recognition site for the binding of the DnaA protein, which is involved in the initiation of bacterial DNA replication.
When DnaA proteins are in their ATP-bound form, what do they do?
Bind to the five DnaA boxes in oriC to initiate DNA replication.
DnaA proteins also do what?
Bind to each other to form a complex.
What are other DNA-binding proteins that help DnaA proteins?
HU
IHF
What does DnaA result in?
It bends around the complex of DnaA proteins and results in the separation of the At-rich region.
Why is the AT-rich region easy to separate?
Because AT base pairs only have two hydrogen bonds while GC has three.
How many replication forks are formed at the origin?
Two
What happens after separation of the AT-rich region?
The DnaA proteins, with the help of DnaC proteins, recruit DNA helicase proteins.
What are DNA helicase proteins?
An enzyme that separates the two strands of DNA.
What is another name for DNA helicase?
DnaB protein
What happens when DNA helicase encounters a double-stranded region?
It breaks the hydrogen bonds between the two strans, thereby generating two single strands.
How do DNA helicases separate the two strands?
Two DNA helicases start at teh oriC region and continue to separate the DNA strands. They use the energy from ATP hydrolysis to catalyze the separation of the double-stranded parental DNA. DNA hlicases bind to single stranded DNA and travel along the DNA in a 5’ to 3’ direction to keep the replication fork moving.
It promotes the movement of two replication forks outward from oriC in opposite direction.
What is bidirectional replication?
The phenomenon in which two DNA replication forks emanate in both directions from an origin of replication.
The GATC methylation sites within oriC are involved in what?
Regulating DNA replication.
GATC sites are methylated by what?
An enzyme known as DNA adenine methyltransferase (Dam).
Prior to DNA replication, the GATC sites are what?
Methylated in both strands. Which facilitates the initiation of DNA replication at the origin.
Following DNA replication, the newly made strands are not what?
Methylated, because adenine rather than methyladenine is incorporated into the daughter strands.
The initiation of DNA replication at the origin does not readily occur until what?
After it has become fully methylated.
Why does DNA replication not occur again too quickly?
It takes several minutes for Dam to methylate the GATC sites within the region.
A site in a chromosome where DNA replication begins is
a. a promoter
b. an origin of replication
c. an operator
d. a replication fork.
b. an origin of replication
The origin of replication in E. coli contains
a. an AT-rich region
b. DnaA box sequences
c. GATC methylation sites.
d. all of the above.
d. all of the above.
What is the function of DnaA proteins?
Bind to DnaA box sequences within the origin to initiate DNA replication
What is the function of DnaC proteins?
Aid DnaA in the recruitment of DNA helicase to the origin.
What is the function of DNA helicase (DnaB)
Separates the double-stranded DNA
What is the function of Topoisomerase II (DNA gyrase)?
Removes positive supercoiling ahead of the replication fork
What is single-strand binding proteins?
Bind to single-stranded DNA and prevent it from re-forming a double-stranded structure.
What is the function of Primase?
Synthesizes short RNA primers.
What is the function of DNA polymerase III?
Synthesizes DNA in the leading and lagging strands.
What is the function of DNA polymerase I?
Removes RNA primers, fills in gaps with DNA
What is the function of DNA ligase?
Covalently attaches adjacent Okazaki fragments.
What is the function of Tus?
Binds to ter sequences and prevents the advancement of the replication fork.
Why is primase needed for DNA replication?
DNA polymerase cannot initiate DNA replication on a bare template strand.
How does unwinding of the helix occur?
DNA helicase breaks the hydrogen bonds between the base pairs and thereby unwinds the strands. This action generates positive supercoiling ahead of each replication fork. Topoisomerase II travels in front of DNA helicase and alleviates positive supercoiling.
After the two parental DNA strands have been separated and the supercoiling relaxed, what must happen?
The strands must be kept separated until the complementary daughter strands have been made. This is done by single-strand binding proteins. They bind to the strands of parental DNA and prevent them from re-forming a double helix. The bases within the parental strands are kept in an exposed condition that enables them to hydrogen bond with individual nucleotides.
What happens during DNA replication?
Starts with the synthesis of short strands of RNA called RNA primers. These strands of RNA are synthesized by the linkage of ribonucleotides via an enzyme known as primase. This enzyme synthesizes short strands of RNA usually 10-12 nucleotides, and these strands prime the process of DNA replication.
What is the leading strand?
A strand during DNA replication that is synthesized continusouly toward the replication fork.
What is the lagging strand?
A strand during DNA replication that is synthesized as short Okazaki fragments in the direction away from the replication fork.
What happens in the leading strand?
A single primer is made at the origin of replication.
What happens in the lagging strand?
Multiple primers are made.
What happens during the synthesis of DNA?
DNA polymerase catalyzes the formation of covalent bonds between adjacent nucleotides and thereby makes the new daugther strands.
In E coli, what five proteins function as DNA polymerase?
Polymerase I Polymerase II Polymerase II Polymerase IV Polymerase V
In E coli, Polymerases I and III are involved in what?
Normal DNA replication
In E coli, Polymerases II, IV and V are involved in what?
DNA repair and replication of damaged DNA.
DNA polymerase III is responsible for what?
Most of the DNA replication because it is a large enzyme consisting of 10 different subunits that play various roles in the DNA replication process.
The complex of all 10 subunits of Polymerase III is called what?
DNA polymerase III holoenzyme
What are the subunits of Polymerase III?
Alpha Epsilon Theta Beta Tau, gamma, delta, delta', Psi, Chi
What is the function of the subunit alpha in polymerase III?
Catalyses the bond formation between adjacent nucleotides and synthesizes DNA
What is the function of the subunit Epsilon in polymerase III?
3’ to 5’ proofreading (removes mismatched nucleotides).
What is the function of the subunit theta in polymerase III?
Accessory protein that stimulates the proofreading function.
What is the function of the subunit beta in polymerase III?
Clamp protein, which allows DNA polymerase to slide along the DNA without falling off.
What are the functions of the subunits Tau, gamma, Delta, Delta’, Psi, Chi in polymerase III?
Clamp loader complex, involved with helping the clamp protein bind to the DNA.
What are the common structural features of DNA polymerases in both bacterial species?
The catalytic subunit of all DNA polymerases has a strcutre resembling a human hand.
What common procedure is seen with DNA polymerases in all bacterial species?
The template DNA is threaded through the palm of the hand; the thumb and fingers are wrapped around the DNA. The incoming deoxyribonucleoside triphosphates (dNTPs) enter the catalytic site, bind to the template strand according to the AT/GC rule, and then are covalently attached to the 3’ end of the growing strand. DNA polymerase also contains a 3’ exonuclease site that removes mismatched bases.
Is the template strand read in the 5’ to 3’ or the 3’ to 5’ direction?
3’ to 5’
What are the two unusual features of DNA polymerase?
It cannot begin DNA synthesis by linking together the first two individual nucleotides. Rather the enzyme can elongate only a preexisting strand starting with an RNA primer or existing DNA strand.
Second, the directionality of strand synthesis can only occur in the 5’ to 3’ direction.
What is the result of the two unusual features of Polymerase III?
The synthesis of the leading and lagging strands show distinctive differences.
How does the synthesis of the leading strand occur?
DNA polymerase III catalyzes the attachment of nucleotides to the 3’ end of each primer, in a 5’ to 3’ direction. One RNA primer is made at the origin, and then DNA polymerase III attaches nucleotides in a 5’ to 3’ direction as it slides twoard the opening of the replication fork. The synthesis of the leading strand is continuous.
How does the synthesis of the lagging strand occur?
The synthesis of DNA also elongates in a 5’ to 3’ manner, but it does so in the direction away from the replication fork. RNA primers repeatedly initiate the synthesis of short segments of DNA; the synthesis is discontinuous.
In bacteria, how long are the fragments added on the lagging strand?
1000-2000 nucleotides.