Chapter 12 - The replicon: Initiation of Replication

Question 1

A replicated region appears as

Answer 1

A replication bubble within nonreplicated DNA.

Question 2

Replication is initiated at

Answer 2

A specific sequence/region called the Origin.

Question 3

Replicon

Answer 3

The DNA that is duplicated with initiation of

replication at the origin.

Question 4

Replication unit(s) on a prokaryotic cell

Answer 4

One replicon

Question 5

Replication unit(s) on a eukaryotic cell

Answer 5

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.

Question 6

Number of replication initiation events that occur for every cell division in prokaryotes or eukaryotes

Answer 6

A single replication initiation event occurs at the origin

once for every cell division.

Question 7

In eukaryotes what is duplicated during Gap 1 (G1)

Answer 7

RNA, protein, lipids, carbs, etc.

Question 8

In eukaryotes what is duplicated during Synthesis (S)

Answer 8

DNA ONLY

Question 9

Where is/are the checkpoint(s) for damaged DNA

Answer 9

End of Gap 1, end of Gap 2

Question 10

Where is/are the checkpoint(s) for chromosome misalignment

Answer 10

End of M

Question 11

Single replication fork

Answer 11

Replication is unidirectional when a single replication fork is created at an origin.

Question 12

Two replication forks

Answer 12

Replication is bidirectional when an origin creates two replication forks that move in opposite directions. Most prokaryotes and eukaryotes use bidirectional replication.

Question 13

θ structure

Answer 13

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.

Question 14

What happens if replicating chromosomes become catenated?

Answer 14

Chromosomes require segregation.

Question 15

The origin of E. coli

Answer 15

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.

Question 16

What is the Xer site-specific recombination system?

Answer 16

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.

Question 17

Where are a majority of genes transcribed?

Answer 17

Leading strand.

Question 18

What happens when the replication fork encounters a DNA-bound protein or transcription machinery in the SAME direction?

Answer 18

The replication fork can slow down and wait for RNAP to reach transcription termination.
-OR-
Replication fork can bypass the RNAP through unknown mechanisms.

Question 19

What happens when the replication fork encounters a DNA-bound protein or transcription machinery in the OPPOSITE direction?

Answer 19

Both processes are halted and RNAP is displaced.

Question 20

Dam methylase

Answer 20

Converts hemimethylated DNA to full methylated strands. Releases SeqA from the origin.
Methylates the Adenine in the GATC/CTAG palindromic sequence.

Question 21

DnaA

Answer 21

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.

Question 22

At the initiation of replication is the palindromic sequence methylated on one or both strands?

Answer 22

Both strands.

Question 23

In E. Coli does replication generate hemimethylated or fully methylated DNA?

Answer 23

Hemimethylated - cannot initiate replication.

Question 24

SeqA

Answer 24

Binds hemimethylated DNA; delays re-replication by sequestration.
A sequestration factor. Inhibits DnaA binding.

Question 25

Function of DNA methylation in bacterial DNA repair

Answer 25

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.

Question 26

Initiation at oriC requires what 6 proteins to form a protein complex on the membrane?

Answer 26

1. DnaA 2. DnaB 3. DnaC 4. HU 5. Gyrase 6. SSB

Question 27

DnaB

Answer 27

Helicase that unwinds and extends the open region. Activated following release of DnaC. Begins replication elongation.

Question 28

Gyrase and SSB

Answer 28

Support the unwinding reaction occurring at oriC.

Question 29

DnaG

Answer 29

Primase; Releases DnaC.

Question 30

HU

Answer 30

Support open structure at OriC.

Question 31

Multiple replicons in eukaryotes

Answer 31

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.

Question 32

Origin of S. Cerevisiea (a eukaryote)

Answer 32

``` 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) ```

Question 33

Autonomously replicating sequence (ARS)

Answer 33

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)

Question 34

ORC (origin recognition complex)

Answer 34

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.

Question 35

Licensing factor

Answer 35

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.

Question 36

Cdc6

Answer 36

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.

Question 37

Pre-replication complex in eukaryotes

Answer 37

Cdc6, CdtI, MCM proteins bound to ORC

Question 38

Post-replication complex in eukaryotes

Answer 38

When replication is initiation, Cdc6, Cdt1, MCM proteins are displaced. Origin only includes ORC.

Question 39

MCM 2,3,5

Answer 39

Regulatory components

Question 40

MCM4,6,7

Answer 40

Helicase components