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What are the structural features of DNA?
- Double Helix
- Nucleotides
- Four Bases
- Hydrogen Bonding between bases
- A-T & G-C
- Antiparallel alignment
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List and describe the methods of replication of DNA.
- Dispersive : Fragments of the original are dispersed throughout the new DNA strand.
- Conservative : New DNA strand contains none of the original DNA.
- Semi-Conservative : Each Helix of DNA has one strand from the original DNA.
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Describe the direction of synthesis of bacterial daughter DNA.
It is bidirectional.
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What is the origin of replication called in E. Coli?
oriC (Origin on Chromosomal Replication)
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What are the three types of DNA sequences in the oriC?
- AT rich region
- DnaA box (5)
- GATC Methylation Site
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What binds to the five DnaA boxes?
DnaA protein
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What takes place after DnaA proteins bind to the five DnaA boxes?
Stimulation occurs, which causes cooperative binding of additional 20-40 DnaA proteins to form a large complex.
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What causes the AT-rich region to seperate in initiation of replication?
Tension of DNA being wrapped around DnaA protein complexes.
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Following denaturation of AT-rich regions in bacterial DNA replication, what promotes helicase enzymes (DnaB helicase) to begin seperation within oriC?
- DnaA proteins
- DnaC proteins
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Does the action of breaking Hydrogen Bonds by DnaB helicase in bacterial DNA replication require energy?
- Yes
- It hydrolyzes ATP for energy.
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What enzyme alleviates supercoiling in the strand as helicase splits apart DNA in bacterial DNA replication?
- DNA gyrase
- (Topoisomerase)
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What is the function of SSB proteins in bacterial DNA replication?
These bind to the strands of parental DNA and prevent them from reforming a double helix until complementary daughter strands can be formed.
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What enzyme synthesizes the leading strand of DNA?
DNA polymerase 3
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Describe the proces of DNA replication of the lagging strand in bacterial DNA replication.
- Primase synthesizes RNA primer so that Polymerase 3 can bind to the primer and begin.
- Polymerase 3 elongates the primer (producing an okazaki fragment). Then leaves strand.
- Polymerase 1 excises RNA primer and fills the gap.
- DNA ligase covalently links the okazaki fragments to form continuous strand.
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What type of bond is formed between nucleotides by DNA polymerases in daughter strands of bacterial DNA replication?
Covalent bonds
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What DNA polymerases are involved in normal DNA replication in bacterial DNA replication?
- DNA polymerase 1
- DNA polymerase 3
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Describe the structure and function of DNA polymerase 1.
- Single subunit
- Fills in small regions where RNA primers were located.
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Describe the sructure and function of DNA polymerase 3.
- 10 subunits
- Beta clamp : Wraps the polymerase around the strand to lock the enzyme onto the strand.
- Alpha : Synthesizes DNA.
- Theata and epsilon : Proofread nucleotides.
- It is responsible for most of DNA replication.
- A processive enzyme.
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What is a holoenzyme?
A multi-subunit enzyme.
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What are the two oddities of DNA polymerase in bacterial cells?
- Unable to initiate DNA synthesis by linking togehter two individual nucleases.
- Is not bidirectionally synthetic.
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Where are TER sequences located on the bacterial chromsome?
Opposite of the oriC.
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What binds to the TER sequences to stop the replication fork?
Termination utilization substance (TUS) proteins
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What is the function of T1 and T2 TER sequences in the bacterial chromosome?
- T1 : allows advancement of clockwise moving forks, but prevents the advancement of counterclockwise moving forks.
- T2 : allows advancement of counterclockwise moving forks, but prevents the advancement of clockwise moving forks.
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What links the two daughter strands creating two circular double stranded molecules in bacterial DNA replication?
DNA Ligase
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What is a Cantenane?
Two interlocked circular DNA strands.
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In E. Coli, what breaks cantenanes and rejoins the unlocked DNA?
Topoisomerases
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What is a Primosome?
DNA helicase + Primase
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What is a Replisome?
Primosome + two DNA polymerases holoenzymes
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What is Dimeric DNA polymerase?
Two polymerase holoenzymes that move as a unit toward the replication fork.
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What are the reasons that DNA replication exhibits a high degree of accuracy?
- Hydrogen bonds betwen correct nucleotide partners are more stable.
- Exonucleases remove mismatch bases at 3' end of a newly made strand.
- Subunit epsilon, alpha, and theata identify and remove mismatched nucleotides in the 3' to 5' direction.
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What are the two different initiation mechanisms in bacterial cells?
- 1) Insufficient amount of DnaA proteins to bind to all DnaA boxes within the oriC, thus preventing premature replication.
- 2) Temporary lack of adenine methylation in the GATC sites within the oriC. Dam enzymes take several minutes to methylate the DNA for replication to start.
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What does the term Hemimethylation refer to?
- Methylation on one strand.
- DNA must be full methylated in bacterial cells for replication to begin.
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What replication enzymes do eukaryotes share with prokaryotes?
- Helicase
- Primase
- Ligase (Topoisomerase)
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How many Origins of replication do eukaryotes have?
Numerous
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Describe the direction of replication of DNA in eukaryotes.
- It is bidirectional from many origins of replication.
- Takes place in S phase.
- Forks eventually meet and complete the process.
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What are ARS elements?
- Autonomously replicating sequences
- 50bp
- High AT content
- ATTAT (A or G) TTTA
- Equivilent to AT-rich area of bacteria
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What is the ORC?
- Origin of recognition complex
- Six subunit protein complex that acts as the initiator of eukaryotic DNA replication when it binds to eukaryotic DNA replication when it binds to ARS during G1 phase.
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How many different DNA polymerases are present in a eukaryotic cell?
12
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Where can DNA polymerase enzymes be found in a eukaryotic cell?
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What do bacteria and Eukaryotes use to remove RNA primers?
- Bacteria : DNA Polymerase 1
- Eukaryote : Flap endonuclease
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When does the synthesis of new histone proteins take place?
S phase
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Describe the despersion of histone proteins between daughter strands of DNA.
Each strand contains a random mixture of original and newly assembled octamers.
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What is a Telomere?
Moderatly repetitive random array with a 3' overhang region (12-16 nucleotides)
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Describe the composition of Telomerase.
- It is a Ribonucleoprotein.
- Contains both proteins and RNA (complementary to the telomeric repeats at the end of the chromosomes).
- Its RNA molecule is calle TERC (Telomere RNA Component)
- Its protein component is called TERT (Telomere Reverse Transcriptase)
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Describe the function of Telomerase.
is a reverse transcriptase that synthesizes DNA from an RNA template.
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What is polymerization?
Attachment of a series of six-nucleotide sequences (tandom repeats) to the end of a DNA strand.
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Is Telomerase found in all eukaryotic cells?
- No.
- It is only found in few somatic cells.
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