Lecture RRL1. DNA replication Flashcards
What is the DNA helicases
They are enzymes that are used to separate the two strands
What are the main characteristics of DNA polymerization
- it is catalyzed by a DNA polymerase
- requires deoxynucleoside 5’ - triphosphates (dNTPs, where N = A, T, C or G )
- requires a primer (can be DNA or RNA)
- proceeds from a 5’ to 3’ direction
What is the role of the topoisomerase
It relieves the supercoils, generated by torsion as the helicase proceeds to separate the strands
TRUE or FALSE, Okazaki fragments form on the leading strand
FALSE, Okazaki fragments form on the lagging strand
What is an Okazaki fragment?
Okazaki fragments are short and discontinuous fragments consisted of DNA and RNA
What is the role of the DNA ligase?
its ligates the two adjoining DNA molecules that have replaced the RNA components of the Okazaki fragments
On which strand is the DNA ligase mainly kept
Lagging strand, it is also present on the leading strand but it only stays there for a short time ( it does it’s thing and dips super quickly)
what are the three complexes forming the CMG helicase
- Hexamer of MCM proteins ( ring, it needs the other 2 accessory complexes to work, it cannot work on it’s own, BUT it is the complex that does the actual action of the helicase )
- Cdc45 complex
- GINS complex
To which strand does the CMG helicase bind
leading
What is the role of the Replication Protein A (RPA)
- It is very abundant in the nucleus
- (binds single-stranded DNA )
- Keep single-strand DNA in a format where the DNA polymerase can use it (it extends the bases)
- prevents the formation of secondary structures of the single-stranded DNA
What are the 3 polymerases working on the replication fork ?
- epsilon
- delta
- alpha (which also works as a primase)
What is the role of the polymerase epsilon?
- carries out leading strand DNA synthasis
What the processivity factor in eucaryotes named?
PCNA
What is a PCNA?
- It is a processivity factor in eucaryotes
- it is a homotrimeric (homotrimer = composed of 3 identical proteins) protein
- it forms a circle around the double-stranded DNA
What is the role of PCNA?
keeping the polymerases working on the strand for longer, so preventing the detachment of polymerases
epsilon and delta
What is the role of pol delta?
- it replaces the pol alpha/primase complex
- completes the synthesis of an Okazaki fragment
( it works with a PCNA complex, just like pol epsilon)
What are the 2 functions of polymerase alpha?
- DNA polymerase
- Primase
it generates a primer and then extends it to form a hybrid that contains RNA and DNA (that is eventually going to be removed from the lagging strand by the polymerase delta)
Since PCNA is a ring, how does it attach to the DNA strand?
What loads the PCNA is the RFC complex (clam loader), it opens the PCNA so that it can attach around the strand.
What are the enzymes (4 total) involved in the removal of the RNA primer? And what do they do?
- ribonuclease H (recognizes RNA/DNA hybrids + degrades the RNA part)
- FEN-1 ( displaces RNA molecule to the 5’ end of Okazaki fragments, so that it can be degraded more easily)
- pol delta ( replaces the RNA with DNA)
- DNA ligase ( ligases the DNA fragments)
what base pairs are found at replication origins? and why?
A (adenine) T (thymine) pairs
because they only have 2 hydrogen bonds which is easier to break
T or F, Human cells have a single origin of replication
FALSE, the genome has tens of thousands of them
what drives the unwinding by the CMG helicase?
ATP hydrolysis
What is the role of the ORC (Origin Recognition Complex)
- mark replication origins
- load helicase (aided by other initiation factors)
what is the next step following the unwinding?
( last step of unwinding) Leading-strand primer synthesis, RPA binds to single-stranded regions
( next step) Primer synthesis
