Ch. 20: DNA Replication Flashcards
Semiconservative replication
When a new double-stranded DNA molecule is formed, one strand will be from the original template, and one will be newly synthesized
Who discovered semiconservative replication?
Meselson and Stahl
Is DNA replication unidirectional or bidirectional?
Bidirectional
Bidirectional DNA replication
The parent strands of DNA are antiparallel and can only be synthesized in the 5’ to 3’ direction, so the two new strands are synthesized in opposite directions
Leading strand synthesis
Occurs in the 5’ to 3’ direction
Lagging strand synthesis
5’ to 3’ direction, but in a discontinuous manner (Okazaki fragments)
What seals together the Okazaki fragments?
DNA ligase
What opens up the DNA helix?
Helicase
Replication fork
The point at which the DNA helix is separated into two strands to allow for the replication of both strands
DNA polymerase mechanism
Responsible for the bulk of DNA synthesis
What does DNA polymerase do?
Catalyzes the addition of a deoxynucleotide (dNTP) to the 3’ end of the growing strand
Klenow fragment
Contains polymerizing and editing modes without the 5’ to 3’ exonuclease activity
Cleavage of DNA polymerase 1 results in what two fragments?
- 5’ to 3’ exonuclease activity
- Klenow fragment
5’ to 3’ exonulcease activity
Makes is unsuitable for many applications
What does the E. coli replication fork consist of?
- Pol III
- Helicase
- Primase
- Gyrase
- Single-stranded DNA binding proteins
Tethering both Pol III core complexes to helices by T subunits in the beta-clamp loading complex…
Ensures that the leading and lagging strand synthesis occur at a similar rate
Clamp loader complex
Mediates the coordinated DNA synthesis on the leading and lagging strand templates by 2 Pol III core complexes
Pol III Core
On the lagging strand template, it alternates between bound and unbound forms as each Okazaki fragment is made
Termination Proteins (Prokaryotes)
To initiate replication, DNA synthesis at Oric must be separated. It is defined by sequence; several steps involve ATP binding or hydrolysis
Termination region
DNA synthesis is initiated at Oric and proceeds bidirectionally until the DNA replication forks each reach halfway around the genome at this sequence
Formation of the pre-replication complex (pre-RC)
Starts with the binding of CdC6, CdH, and two McM2-7 helices to the ORC
When are the pre-RC’s formed?
During the G1 phase
When does the activation of specific sites of the pre-RC happen?
During the S phase
What does the pre-RC do?
It converts to a replisome progression complex (RPC) after the binding of additional proteins at the two replication forks.
Short patch-repair (base excision repair)
Involves removal and replacement of a single nucleotide catalyzed by DNA polymerase and DNA lyase
Long patch repair (base excision repair)
Involves the synthesis of up to 10 or more nucleotides using a strand displacement mechanism; Flap endonuclease (FEN) removes the displaced strand, leaving behind a single-strand nick that is sealed by DNA ligase
Nucleotide excision repair
Initiated by recognition of the lesion by UvrAB complex; used for large lesions that distort the helical nature of DNA
What causes nicks in the DNA in nucleotide excision repair?
UvrB and UvrC
What does UvrD do in nucleotide excision repair?
Removes the polynucleotide containing the lesion, followed by gap repair using Pol I and ligase
What does UvrABC exonuclease do in nucleotide excision base repair?
Scans for errors in the DNA
DNA Photolyase (Direct Repair)
Photolyase converts thymine dimers to their normal structures via a 6 step mechanism
6 Step Mechanism of DNA Photolyase
- Absorption of lightt by the folate coenzyme (MTHF )
- Energy transfer from the MTHF* to FADH- to generate the excited state *FADH-
- E- transfer from *FADH- to the cyclobutane dimer substrate
- Radical reaction breaks the 1st bond
- Radical reaction breaks 2nd bond
- E- is transferred to FADH, and adjacent pyrimidines restored to normal structure
MGMT (Direct repair)
The MGMT enzyme uses a suicide mechanism to remove the CH3 group from O6-methylguanine
An active site cytosine residue displaces the methyl group to produce a homocysteine residue
This modification inactivates the protein, which is then degraded to metabolize the homocysteine
Repair of Single-Strand breaks in eukaryotes
The process of repairing a single-strand DNA break in eukaryotes is very similar to base excision repair and nucleotide excision repair mechanisms
What is the difference in single-strand breaks in eukaryotes vs. prokaryotes?
Different repair proteins are required for short repair and long repair
How is the gap fixed after long and short DNA repair (eukaryotes)?
It is sealed by one of several DNA ligases (Lig1 or Lig3)
What are the two methods of repair for double-strand breaks in eukaryotes?
- Homologous recombination
- Nonhomologous end joining
Non-homologous end joining
- Does not require a DNA template strand
Homologous recombination (Double-strand break repair)
In this repair method, a DNA template is used to fix the double strand break through homologous combination. This is a method used in CRISPR.
