replication Flashcards
direction of replication
¥ DNA synthesis occurs in a 5’ -> 3’ direction by formation of phosphodiester bonds
explain the use of primers where they are added, how they are added
- the 3’ hydroxyl at the end of the growing DNA strand makes a nucleophilic attack on the phosphate of the incoming nucleoside triphosphate (dNTP)
- form a diester bond.
- releases pyrophosphate (two phosphate atoms)
how is does helices work
• DNA Helicase uses ATP to separate parental DNA strands at the Replication Fork and move the Replication Fork forward
Helicase continually uses ATP binding and hydrolysis to drive DNA strand separation and progression of the replication fork
what is Werner Syndrome
The WRN gene in humans encodes for a variant of DNA helicase, the RecQ-like type 3 helicase
known to cause Werner syndrome that causes premature ageing.
result in an abnormally shortened Werner protein (i.e. helicase).
Evidence suggests that the mutant helicase is not transported into the cell nucleus
The truncated helicase may also be broken down too quickly, leading to a loss of helicase in the cell.
Without normal Werner protein/helicase in the nucleus, cells cannot perform the tasks of DNA replication
how do you increase the processivity of DNA Polymerases
association with a Sliding Clamp
how do you increase the processivity of DNA Polymerases
association with a Sliding Clamp
nonprocessive DNA polymerase adds nucleotides at a rate of one nucleotide per second Processive DNA polymerases however, add multiple nucleotides per second, drastically increasing the rate of DNA synthesis.
what is the sliding clamp?
once DNA primase has done its job, two further essential factors act to complete replisome assembly.
These are the clamp loader (i.e. replication factor C) and the sliding clamp (i.e. PCNA).
The clamp load use ATP hydrolysis to lead the sliding clamp onto the DNA, near the primer:template junction.
The sliding clamp is doughnut shaped and the clip is locked onto the DNA once it is threaded through.
The loading of the sliding clamp displaces primase and triggers recruitment of DNA polymerase δ and ε
With the sliding clamp loaded, DNA is reeled through the replisome and polymerase ε synthesises DNA continuously on the leading strand. On the lagging strand DNA polymerase δ synthesises DNA in bursts
what is Single-stranded DNA Binding Proteins and what is its job
as helicase unwinds double-strand DNA, its creating sections of single-strand DNA in both leading and lagging strands
the single-stranded DNA can anneal to itself due to complimentary DNA sequences and create DNA hairpins
hairpins will impede to progress of DNA polymerase and compromise its processivity
To prevent this problem, SSBs decorate single-stranded DNA like beads on a string to stop the formation of DNA hairpins
aids the processivity
what are DNA topoisomerases? there role
DNA topoisomerases prevent DNA from becoming tangled during DNA replication and enhance processivity of DNA polymerase
¥ Helicase unwinding of parental DNA strands at the Replication Fork introduces superhelical tension into the DNA Helix.
¥ Tension is relaxed by DNA Topoisomerases, which nick and reseal the backbone of the parental helix
difference between type 1 and 2 topoisomerases
¥ Type I Topoisomerases nick and reseal one of the 2 DNA strands, no ATP required
¥ Type II Topoisomerases nick and reseal both DNA strands, ATP required
what is the replisome
DNA replication machinery
Initiation of DNA replication in eukaryotes is biphasic:
what are the two steps and when do they occur
- Replicator Selection occurs in G1 phase - formation of a pre-Replicative Complex
- Origin Activation occurs in S phase - unwinding of DNA and recruitment of DNA Polymerase
the steps required for the formation of a Pre-Replicative Complex
• Origin Recognition Complex (ORC) binds to Replicator sequence, i.e. ARS sequence in yeast
Helicase-loading proteins Cdc6 and Cdt1 bind to ORC
The Helicase Mcm2-7 binds to complete formation of pre-RC
ORC- Cdc6 - Cdt1 uses ATP hydrolysis to crack open the two hexameric helicase MCM2-7 rings so they can wrap around DNA in a head-to-head orientation
At this point, we have assembly of a pre-Replicative complex in G1. And nothing else happens until S-phase
how is the Pre-RC activated in S phase
High levels of Cyclin-dependent kinase (Cdk) activity in S-phase activates existing pre-RC but prevents formation of new pre-RCs
in S-phase, Cdks activate by phosphorylation a pre-assembled pre-replicative complex
