(Dr. Heinemann) (Unit C) Topic 15 Flashcards
Where does DNA replication start?
Replication origins
How many replication origins do organisms use?
- Prokaryotes usually have one
- Eukaryotes have multiple
What are characteristics of replication origins?
Rich in AT base pairs
* Easier to pull apart than CG base pairs
Initiator proteins
Bind to sequences at replication origins and pull them apart
Are initiators present in eukaryotes?
Yes, they are present in eukaryotes and prokaryotes
Helicase
(Prokaryotes)
Binds after initiator proteins pull chains apart
* Consumes ATP to unwind DNA during the replication process
Helicase
(Eukaryotes)
Inactive helicase is part of pre-initiation complex that binds to each replication origin
* Upon activation, starts separating strands at each origin
What is the replication bubble?
The region which the DNA has become single-stranded
Single-strand binding proteins (SSBs)
Associates with unwound regions and prevents them from re-forming base pairs
Replication forks
Area where the double stranded DNA becomes single stranded
True or False:
DNA polymerase synthesizes DNA by directly using the template strand
False, it needs something to attach nucleotides to
Primase
Synthesizes short RNA primers on the separated DNA strands for DNA polymerase to work on
Primers
* How long are they?
* In what direction are they synthesized?
- ~10 nucleotides
- 5’ to 3’ direction
After primer synthesis, what protein is loaded onto the primer-template complex?
Sliding clamp protein
What is the goal of the sliding clamp protein?
It will not spontaneously dissociate from douoble-stranded DNA
* Keeps DNA polymerase on the chain
What does DNA polymerase do?
Synthesize most of the new DNA
What direction does DNA polymerase synthesize things in?
5’ to 3’
* Extends the primer from its 3’ end
What is replicative DNA polymerase known as in prokaryotes?
DNA polymerase III
When complementary bases are added by DNA polymerase, what is the by-product?
Pyrophosphate (PPi)
* Hydrolysis of ATP to AMP + PPi
Why is DNA polymerase called a “processive” enzyme?
Undergoes several rounds of catalysis before dissociating from the template
Replisome
The complex of proteins that carries out replication at a replication fork
Leading strand
Synthesis on this strand is said to be continuous
* DNA synthesis can continue for long distances (usually about 5 * 10^5 using only one primer)
Why does the DNA synthesis on the complementary template strand occur in the direction opposite the which the replication fork is moving?
DNA is anti-parallel
What does discontinuous synthesis of DNA on the lagging strand form?
Okazaki fragments
What are Okazaki fragments connected by?
DNA ligase
Okazaki fragments
* Length in prokaryotes
* Length in eukaryotes
- 500-2000 nucleotides long
- 1/10th of the length in prokaryotes
What is the traditional model for explaining the replication on leading and lagging strands occurring concurrently?
“Trombone” model
What is the present explanation for efficiency of Okazaki fragment synthesis?
3 DNA polymerase complexes can associate with the helicase simultaneously
Does DNA polymerase have proofreading ability?
Human DNA polymerases have proofreading ability
* They are very accurate without proofreading, but it makes it more accurate
What kind of activity does DNA polymerase have to proofread?
3’ to 5’ exonuclease activity
* Can remove mononucleotides from the 3’ end of a DNA strand
What is the accuracy of DNA replication with proofreading?
1 error/10^6 or 10^7 bases
In the Okazaki fragment, when does DNA polymerase III dissociate?
(Only in prokaryotes)
When it encounters the RNA primer from the previously synthesized fragment
DNA polymerase I
(Only in prokaryotes)
- Degrades RNA primer
- Synthesizes DNA
What activity does DNA polymerase I have?
(Only in prokaryotes)
5’ to 3’ exonuclease activity
* Degrades the RNA primer while synthesizing DNA
What does DNA polymerase I synthesizing DNA result in?
(Only in prokaryotes)
“Nick translation”
What does DNA ligase require to work?
- In bacteria: NAD+
- In humans: ATP (hydrolyzes to AMP + PPi)
What does DNA ligase require on the DNA strands?
- 3’ hydroxyl
- 5’ phosphate
How does Okazaki fragment maturation in eukaryotes work?
- Replicating DNA polymerase does not dissociate from the template
- RNA primer is removed by another enzyme
What is the RNA primer removed by eukaryotes?
Examples include:
* RNase H
* Flap endonuclease (removes ssRNA that have been pushed aside by DNA polymerase)
What are telomeres?
Protein-DNA structures found at the ends of chromosomes
Describe:
Human telomeres
- 2000-10000 base pair double-stranded region containing many repeats of a 6-nucleotide sequence (TTAGGG)
- Followed by 100-300 nucleotide 3’ overhand that loops back and displaces an earlier section of the same strand
What are the two main functions of telomeres?
- Enable cell to distinguish between the end of the chromosomes vs. double-strand break in the middle of the chromosome
- Replicating the lagging strand at the ends of chromosomes (provides extra DNA so that coding DNA is not lost during replication)
True or False:
Chromosomes get shorter every single time they are synthesized
True
Telomerase
Synthesize and lengthen telomeres
* A type of DNA polymerase
* Uses associated RNA strand as a template
Expression of Telomerase
* Normal
* Abnormal
- Expressed during embryo development and in germ-line cells
- Expressed in adulthood in cancer cells
Azido thymidine (AZT)
Also called zidovudine or Retrovir
* Anti-viral agent used to combat HIV
How does AZT work?
- Taken orally
- Converted to triphosphate form
- Used as a substrate in place of dTTP during DNA synthesis
- New nucleotide cannot be added because it does not have a 3’ hydroxyl group
- Prevents formation of new virus particles
Why is AZT 100 times more effectively on reverse transcriptase than human DNA polymerase?
Viral enzyme (reverse transcriptase) has higher affinity for AZT than for dTTP
Acyclovir
A seletive inhibitor of the herpes virus DNA polymerase
How does Acyclovir work?
Causes chain termination when incorporated into a growing DNA strand
