DNA Replication Flashcards
Origins of DNA Replication in prokary. and eukary.
Origins are the site of initiation of DNA replication
Prokary. : One origin, replication forks move in both direction in a circle
Eukary. : Multiple origins, not all origins fire, tend to occur in clusters, replication forks move in both directions. Dormant origins exist and are only activated if not all DNA is replicated.
Euchromatin
Decondensed/Lightly packed
Available for transcription
Heterochromatin
Condensed/Tightly Packed
Unavailable for transcription
Nucleosome
DNA coiled around a core of histones:
H2A, H2B, H3, and H4 (2 of each)
Nucleosomes affect replication efficiency by:
- inhibiting access of DNA replication machinery to the origin
- slowing down the movement of replication forks
Chromatin remodelling proteins stimulate DNA replication
Roles of Origins of Replication (Essential and non-essential)
- Binding sites for initiator/ origin recognition proteins
- Start site of replication
- Elements for unwinding/distortion of DNA
(Essential)
-Additionally act as binding site for chromatin remodellers (Non-essential, regulatory)
Initial Stages of DNA Replication
- The initiator proteins/origin recognition complexes
bind at or near the origin and recruit DNA helicases to the origin.
-DNA helicases then separate the strands of the DNA,, causing extensive unwinding and providing the replication machinery with access to the ssDNA template.
Initiator Proteins/Origin Recognition Proteins in Prokary. and Eukary.
Prokary. : Initiator DnaA, causes melting of the double helix, exposing a small region of ssDNA (occurs before activation of helicase)
Eukary. : Origin Recognition Complex (ORC) and Cdc6 (ORC loader), bind to and encircle dsDNA at the origin (don’t melt like DnaA). ORC-Cdc6 complex is involved in loading the helicase in an inactive state in G1-phase. Activation occurs later in S-phase.
DNA helicases in prokary. and eukary. (Structure, examples, role, reaction, etc.)
Unwind DNA by disrupting the hydrogen bonds that hold the two strands of DNA together
Their reaction requires NTP hydrolysis
Structure = Hexamer Ring
Prokary. : E.coli, DnaB
Upon binding of DnaC (a helicase loader), DnaB opens. ssDNA produced from melting by DnaA enters the opening in the ring and binds to the central channel of the DnaBC complex. DnaB ring closure and DnaC release leave the DnaB helicase loaded on ssDNA.
Eukary. : Human, MCM complex
ORC and Cdc6 (ORC loader) are encircle the origin duplex DNA. MCM’s 2-7 form the hexamer, with MCM’s 2 and 5 acting as the gate. Cdt1 is a co-loader, and with ORC-Cdc6 is able to load the open ring onto the DNA. Cdc6 and Cdt1 are released upon MCM helicase complex binding.
Single-Stranded DNA Binding Proteins (SSBs) : What are they and what is their role?
SSBs are proteins that bind to ssDNA (as their name implies). They bind cooperatively and without covering access to the bases. They are displaced from the ssDNA as DNA pol. moves in to make the new strand.
Role: SSBs assist in DNA helix-opening processes by:
- Stabilising the unwound single stranded conformation
- Protecting the ssDNA from hydrolysis or formation of hairpin helices
- Facilitating assembly of the replication machinery
- Facilitating primer transfer between polymerases
DNA Replication “Priming”
Synthesis of RNA primer/s by primase.
Provides the 3’ -OH group paired to a DNA template that is required by DNA polymerases to initiate DNA replication.
Primases are small RNA polymerases
In prokary. : E.coli primase is DnaG
In eukary. : Primase is in a complex of 4 proteins
Humans: DNA pol. alpha primase
DNA Replication: Elongation and DNA polymerase in prokary. and eukary.
DNA polymerase extends from the 3’ end of the RNA primer.
DNA polymerase is directed by the template strand.
Accurate DNA replication depends upon correct hydrogen bonding between incoming nucleotides and template.
DNA pol. forms phosphodiester bonds (for the backbone)
DNA pol. has proofreading activity via 3’-5’ exonuclease activity.
Prokary. :
DNA polymerase III (Leading AND lagging strand synthesis, multimer)
DNA polymerase I (monomer) (Repairs damage, also connects Okazaki fragments)
Eukary.:
DNA polymerase alpha/1 (Not processive, no proofreading, only involved in initiation by creating initiator DNA sequence after RNA primer)
DNA polymerase delta/3 (Lagging strand synthesis)
DNA polymerase epsilon/2 (Leading strand synthesis)
DNA Ligase
Catalyses the formation of phosphodiester bonds between 3’OH and 5’P ends of polynucleotides hydrogen bonded to a complementary strand.
Topisomerases
Topoisomerases produce ss or ds transient breaks in the phosphodiester backbone of DNA through formation of covalent protein-DNA intermediates, allowing the DNA to rotate and re-ligate one or both strands to prevent torsional strain.
Processivity in relation to DNA polymerases
Most DNA polymerases have low processivity.
A Clamp is required to increase the processivity of polymerases.
In prokary. : The beta subunit of DNA pol. III acts as a clamp
In eukary. : In humans, the clamp is PCNA (Proliferating cell nuclear antigen)
Clamp loaders:
Prokary. : E.coli gamma subunit complex of DNA pol. III (ATP binding and hydrolysis)
Eukary. : Humans RFC (Replication Factor C) i.e. RFC catalyses the loading of PCNA onto DNA
Telomerase
Adds a telomere repeat sequence to the 3’ end of telomeres.
Telomeres maintained by balance between shortening by replication and restoration by telomerase.
