Chapter 12 - The replicon: Initiation of Replication Flashcards
A replicated region appears as
A replication bubble within nonreplicated DNA.
Replication is initiated at
A specific sequence/region called the Origin.
Replicon
The DNA that is duplicated with initiation of
replication at the origin.
Replication unit(s) on a prokaryotic cell
One replicon
Replication unit(s) on a eukaryotic cell
Multiple replicons
Each replicon is activated at a specific time during the S-phase of the cell cycle (difficult to characterize individual replicons).
Multiple origins of replication ultimately merge during replication.
No termination or ter site required.
Number of replication initiation events that occur for every cell division in prokaryotes or eukaryotes
A single replication initiation event occurs at the origin
once for every cell division.
In eukaryotes what is duplicated during Gap 1 (G1)
RNA, protein, lipids, carbs, etc.
In eukaryotes what is duplicated during Synthesis (S)
DNA ONLY
Where is/are the checkpoint(s) for damaged DNA
End of Gap 1, end of Gap 2
Where is/are the checkpoint(s) for chromosome misalignment
End of M
Single replication fork
Replication is unidirectional when a single replication fork is created at an origin.
Two replication forks
Replication is bidirectional when an origin creates two replication forks that move in opposite directions. Most prokaryotes and eukaryotes use bidirectional replication.
θ structure
In bacteria, the replication bubbles forms the θ structure.
No θ structure in eukaryotes because replication occurs on linear DNA, but eukaryotes do exhibit a replication bubble.
What happens if replicating chromosomes become catenated?
Chromosomes require segregation.
The origin of E. coli
OriC. 245 bp in length.
Contains:
1. Binding sites for the replication initiating protein DnaA.
2. 11 GATC/CTAG palindromic sequences.
3. The Adenine in the sequence is methylated by the Dam
methylase enzyme.
What is the Xer site-specific recombination system?
Includes:
2 Recombinases: XerC and XerD
The dif gene sequence near the replication termination region.
Recombinases bind to the dif site and form Holliday junction.
FtsK, which resolves the junction, is located at the septum.
Replication termination region associates with septum; hence movement of the dif site +/- 30 kb, inhibits recombination event.
Where are a majority of genes transcribed?
Leading strand.
What happens when the replication fork encounters a DNA-bound protein or transcription machinery in the SAME direction?
The replication fork can slow down and wait for RNAP to reach transcription termination.
-OR-
Replication fork can bypass the RNAP through unknown mechanisms.
What happens when the replication fork encounters a DNA-bound protein or transcription machinery in the OPPOSITE direction?
Both processes are halted and RNAP is displaced.
Dam methylase
Converts hemimethylated DNA to full methylated strands. Releases SeqA from the origin.
Methylates the Adenine in the GATC/CTAG palindromic sequence.
DnaA
Replication initiating protein.
The licensing factor (necessary for replication; destroyed after one round of replication).
Active only when bound to ATP.
ATPase activity hydrolyzes ATP to ADP.
Functions to melt DNA.
Binding sites include 13- and 9-mers repeats.
At the initiation of replication is the palindromic sequence methylated on one or both strands?
Both strands.
In E. Coli does replication generate hemimethylated or fully methylated DNA?
Hemimethylated - cannot initiate replication.
SeqA
Binds hemimethylated DNA; delays re-replication by sequestration.
A sequestration factor. Inhibits DnaA binding.
Function of DNA methylation in bacterial DNA repair
Aids the DNA mismatch recognition machinery to decipher between the parental and daughter strand.
The parental strand is the template. Parental strand is the methylated. Daughter strand is unmethylated. Error corrected on the new daughter strand.
Initiation at oriC requires what 6 proteins to form a protein complex on the membrane?
- DnaA
- DnaB
- DnaC
- HU
- Gyrase
- SSB
DnaB
Helicase that unwinds and extends the open region. Activated following release of DnaC. Begins replication elongation.
Gyrase and SSB
Support the unwinding reaction occurring at oriC.
DnaG
Primase; Releases DnaC.
HU
Support open structure at OriC.
Multiple replicons in eukaryotes
Do not function simultaneously.
Each replicon is activated at a specific time during the S-phase of the cell cycle.
Replicons near active genes are replicated first; Replicons in heterochromatin are replicated last.
Replicons near one another are activated at the same time.
Origin of S. Cerevisiea (a eukaryote)
Short sequences called Autonomously Replicating Sequence (ARS). • Common features of ARS: - extends for ~50 bp - 14 bp “core” A domain and additional elements (B domain à replaceable) - 11bp A-T consensus sequence called ARS Consensus Sequence (ACS)
Autonomously replicating sequence (ARS)
Extends for ~50 bp
14 bp “core” (A domain)
Additional elements (B domain(s))
11 bp A-T consensus sequence called ARS Consensus Sequence (ACS)
ORC (origin recognition complex)
Six proteins that bind to an ARS (an origin for replication in yeast) throughout the cell cycle.
Related ORC complexes are found in multicellular eukaryotes.
ORC binding marks the origin site.
The origin site has not been characterized to the same extent as the ARS in yeast.
Licensing factor
Necessary for initiation of replication at each origin. Present in the nucleus prior to replication, but is removed, inactivated, or destroyed by replication.
Initiation of another replication cycle becomes possible only after licensing factor re-enters nucleus or the break down of the nuclear membrane at mitosis.
Cdc6
Key licensing factor; synthesized only in G1 and binds the
ORC.
Cdc6 recruits Cdtl and MCM proteins to bind; this generates the Pre-replication Complex.
Cdc6 degradation prevents reloading of the MCM
proteins onto the origin.
Pre-replication complex in eukaryotes
Cdc6, CdtI, MCM proteins bound to ORC
Post-replication complex in eukaryotes
When replication is initiation, Cdc6, Cdt1, MCM proteins are displaced. Origin only includes ORC.
MCM 2,3,5
Regulatory components
MCM4,6,7
Helicase components
