DNA replication is bi or uni - directional
Bidirectional
How many origins in prokaryotes?
One
How many origins in eukaryotes?
Hundreds
some used more than others
Define semiconservative
Each new strand is one old strand and one new strand.
Replication fork
Site where replication is occurring
What recognizes and binds to origin?
Origin binding proteins
Where are origins likely to be?
AT rich sequences
What unwinds the parental strands?
Helicase
What prevents super coiling?
Topoisomerases in eukaryotes
Gyrase in prokaryotes
What inhibits dna gyrase?
quinolones
what are quinolones?
class of broad spectrum antibiotics
Direction of DNA replication?
5’ —> 3’
What catalyzes the synthesis of DNA?
DNA polymerase
How does DNA polymerase work?
adds deoxyribonucleotides to the 3’- OH of RNA primers then to 3’- OH of growing DNA strands
How many DNA polymerases in prokaryotic DNA replication?
2
Pol I and Poll III
Which is used more? Pol I or Pol III
Poll III
Why is Poll III used more in prok. DNA repl
has a sliding clamp so can work over a longer distance (more processive manner)
What does Pol I do?
Clean-up work during DNA replication and repair
Replaces RNA primers via
5’ - 3’ exonuclease activity
5’ - 3’ DNA polymerase activity
How many polymerases in Eukaryotic DNA replication?
3
Pol alpha
Pol delta
Pol epsilon
What does Pol alpha do?
is a holoenzyme - multi-protein complex
Primase activity
DNA polymerase activity
Synth. first ~20 residues after RNA primer
Does Pol alpha have proofreading activity?
No
What does Pol delta do?
Synthesis of lagging strand
What does Pol epsilon do?
Synthesis of leading strand
Can DNA replication start ‘de novo’?
No. Requires an RNA primer
What prepares the RNA primer?
primase
What does SBB do?
Single Strand Binding protein holds and protects the single strands unwound by helicase during replication
What does helicase do?
unwinds parent DNA strand
What does origin binding protein do?
recognizes and binds to origin
What serves as primer for DNA synth?
RNA
How long is the primer?
~10 residues of RNA
What synthesizes the first ~20 DNA residues onto the primer?
Pol alpha
What synthesizes the leading strand in eukaryotes?
Pol epsilon
What synthesizes the lagging strand in eukaryotes?
Pol delta
What does primase do?
synth the RNA primer
How do the RNA primers get chosen?
use the DNA template
Replication fork?
place where DNA replication is ocurring simultaneously on both parent strands but in opp. directions
e.g. both grow in 5’ - 3’ direction along the growing strand but spatially opposite direction
Which strand grows towards replication fork?
leading strand
Leading strand grows _______________.
continuously
Lagging strand grows _____________.
discontinuously. e.g. okazaki fragments
Which strand grows away from the replication fork?
lagging strand
What are okazaki fragments?
short fragments of DNA formed in synth of lagging strand
how long are okazaki fragments?
Euk: 100-200 bp
Prok: 1000 - 2000 bp
How are RNA primers removed?
5’ - 3’ exonuclease (DNA pol I in e.coli)
How are gaps from RNA primers filled?
with appropriate DNA using template (by pol I in e.coli)
In E. Coli what are the functions of Pol I?
- 5’ - 3’ DNA polymerase activity requiring 3’-OH primer
- 5’ - 3’ exonuclease activity to remove RNA primer
- 3’ - 5’ exonuclease activity for proofreading
How are okazaki fragments joined?
by DNA ligase
What does DNA ligase do?
forms phosphodiester bonds between 3’-OH and 5’-phosphate of two polynucleotide chains to join okazaki fragments
Overall fidelity of DNA replication?
error rate of 10^-9 to 10^-10
2 ways polymerases discriminate between correct and incorrect nucleotide?
- Hydrogen bonding between complimentary nucleotides (A-T, C-G)
- Geometry of the A-T and C-G base pairs that allow them to fit into the active site of polymerase
H-bonding and bp geometry account for what level of accuracy?
1 error in 10,000 to 100,000 correct nucleotieds
If an error occurs, how is it fixed during replication?
Proofreading during replication by 3’-5’ exonuclease activity associated with polymerase complex
Proofreading increases the accuracy of replication by 100 - 1,000 fold. What is the final error rate?
1 error per million to 100 million correct nucleotides
How else can DNA repair occur other than during replication?
Post-replicational repair…see DNA repair lecture
What is synthesis of DNA from RNA?
reverse transcription
Name two instances where reverse transcription takes place?
- Retroviruses
2. Telomerase activity
What does telomerase do?
has reverse transcriptase activity
carries its own RNA template
restores ends of chromosome (telomeres) in human cancer and stem cells
What is the end replication problem?
leading strand can be synthesized to the very end but the lagging strand cannot
Why can the lagging strand NOT be synthesized to the very end?
Need an RNA primer ot begin synth. of each piece.
The last piece has no attachment for primer
What is result of lagging strand not going to the very end of replication?
Telomere gets shorter with each round of replication.
In cancer cells is telomerease turned on or off?
On. (de-repressed) Bad.
blocks normal cell death promoting tumor growth.
In normal cells is telomerase on or off?
Off (repressed)
What is added to the CTD of poll II and what is its function?
The CTD of pol II is phosphorylated. This attaches to the 5’-cap on the growing RNA strand and serves as a “landing pad” for the incoming nucleotides
What protein is a trimer of the PCNA protein and what is its function?
The DNA “sliding clamp”
holds DNA template in the polymerase
aids in processivity
What is the ORC?
Origin recognition complex
