Sept 22 DNA Replication Flashcards
what parts of a nucleotide react when a nucleotide is added to a growing chain?
the alpha phosphate group in an incoming deoxy nucleoside triphosphate (dNTP) reacts with the 3’ hydroxyl group on the growing DNA chain
what does helicase do?
unwinds the duplex DNA
what does topoisomerase do?
relieves supercoils
why are primers needed and how are they made?
DNA polymerases cannot initiate synthesis of a new strand, can only elongate an existing strand
–> therefore a primer is needed
primase forms primer: short RNA molecule complementary to a single stranded region of the unwound duplex DNA
DNA polymerase extends primer to eventually form daughter duplex DNA
what are okazaki fragments?
short discontinuous fragments consisting of RNA and DNA
what does DNA ligase do?
replaces the RNA components of the okazaki fragments with DNA
ligates two adjoining DNA molecules together (in the fragments)
what is the replisome?
molecular machine involved in DNA replication
what are the different parts of the replisome and their functions?
- large T-antigen helicase:
hexamer
helicase
encoded by the viral genome
unwinds double helix at replication fork - Replication Protein A (RPA)
binds single stranded DNA
keeps single stranded DNA template in optimal conformation for DNA polymerase - DNA polymerase epsilon
carries out leading strand DNA synthesis - PCNA
homotrimeric protein
prevents the polymerase epsilon complex from dissociating from the template - primase/polymerase alpha
primase forms the RNA components of the primer and DNA polymerase alpha extends the primer with DNA - Pol alpha/Rfc/PCNA complex
replaces the polymerase alpha/primase complex and completes the synthesis of an okazaki fragment - Rfc/PCNA complex:
RFC is the PCNA loader, opens the PCNA ring and loads it at a primer on DNA
what happens after Ribonuclease H and FEN-1 displace RNA component at the 5’ end of the okazaki fragment
polymerase delta/Rfc/PCNA replaces the RNA with DNA
DNA fragments are the ligated together by DNA ligase
where does replication start?
region called the replication origin
replication origins tend to be AT-rich
what are the steps of DNA replication? (7)
- unwinding
catalysed by the large T-antigen (helicase) driven by the hydrolysis of ATP
RPA binds and stabilises single stranded regions
protects the strands from enzymatic degradation - Primer Synthesis
primase polymerase alpha complexes synthesise primer sequences that are base paired to the parental templates - Extension
pol epsilon/Rfc/PCNA complexes replace the primase Pol alpha complexes and extend the primer sequences - Further Unwinding
further unwinding and binding of RPA to single stranded regions - Further extension
Pol epsilon/Rfc/PCNA complexes continue to synthesise the leading strands - lagging strand primer synthesis
primase pol alpha complexes form primers for lagging strand synthesis - primer extension, primer removal and strand ligation
pol delta/Rfc/PCNA complexes replace the primase pol alpha complexes and extend the primer sequence
removal of the primers is performed by FEN-1 and ribonuclease H
pol alpha/Rfc/PCNA complexes replace the primer sequences with DNA
strands are ligated together by DNA ligase
what are the three DNA polymerases and what do they do?
polymerase epsilon: for the leading strand
polymerase delta: for the lagging strand
polymerase alpha: acts in conjugation with primase
what do DNA polymerases require for the synthesis of a new strand?
- a single stranded DNA template
- a DNA primer base paired with the template and with a free hydroxyl group at the 3’ end to accept a free nucleotide
- a source of dNTPs precursors
why do replication origins tend to be AT-rich?
AT is a double hydrogen bond while CG is a triple hydrogen bond
AT is energetically easier to break than CG
what monomers does primase add?
rNTPs
