Lecture 18: Replication of linear and circular genomes

Question 1

How many strands are there in a replication fork?

Answer 1

Two:
1. Leading strand: Goes forwards (towards
fork
2. Lagging strand: also goes towards fork,
however requires a loop (due to 5’
to 3’ processing)

Question 2

What proteins are involved in a replication fork?

Answer 2

1. Core: where DNA runs through & where DNA is replicated
2. Clamp-loading complex: has a beta-sliding clamp associated with it
3. DnaB opening of DNA strands

Question 3

How many proteins are involved in the initiation of replication in E. coli?

Answer 3

These proteins are involved in finding and opening the correct origin site.

POLYMERASE ISNT ONE OF THEM.

Question 4

How do different proteins interact at initiation?

Answer 4

1. Origin of initiation (oriC) - a region of 
   DNA
2. DnaA-ATP binds to the correct region 
    of oriC
3. This causes this region of oriC to 
    become wound up
4. This causes a different section of oriC 
    called DUE ("DNA Unwinding 
    Element") to undergo separation of its 
    2 strands - referred to as "DnaA- 
    dependent denaturation of the DUE 
    region"
5. This allows different proteins (DnaB) 
    to come and bind to the separated 
    strands of DNA = 2 replication forks
      a. DnaB is bound to DNA via DnaC- 
          ATP, which hydrolyses ATP in a 
          process known as "DnaC- 
          dependent loading of DnaB 
          helicase"

Question 5

What other proteins are involved in initiation?

Answer 5

1. These proteins facilitate DnaA binding to 
   DNA but their mechanisms are unclear:
   a. HU
   b. IHF
   c. FIS

Question 6

What are terminator sequences referred to as? And what do they do?

Answer 6

1. Ter. E.g., TerJ or TerG
2. These are specific ‘termination’
   sequences
3. Some are located within the
   “clockwise fork trap”, others are
   located within the “counter-clockwise
   fork trap”.
4. Two replication forks meet.
   Replication is completed = 4 strands
   of DNA ‘connected’ to each other
   called “catenated chromosomes”
5. DNA topoisomerase 4 (type 2
   topoisomerase) separates the
   chromosomes

Question 7

What is replication like in eukaryotes?

Answer 7

1. Genomes are a lot larger and linear
2. Have “replicons”, meaning regions of
   DNA that are replicated together
   a. 40-100kbp
   b. 1000’s of different replicons
   c. Therefore, 1000’s of different origins to
   be synthesised in a coordinated fashion
   d. Neighbouring replicons usually
   replicated next but not always

Question 8

What is replication initiation like in eukaryotes?

Answer 8

1. Similar way to prokaryotes with oriC,
   but proteins are different and less
   understood
2. Still a helicase (MCM2-7) loaded onto
   DNA
3. Once loaded, it will bring DNA
   polymerase of the appropriate type

Question 9

How does termination take place in eukaryotic chromosomes?

Answer 9

1. No existing 3'- end to begin the    
   growing strand
2. Not possible to copy 3'- ends of linear 
    chromosomes using normal      
    replication apparatus as      
    chromosomes  would gradually 
    shorten
3. "Telomerase"
   a. has RNA subunit and synthesises 
       new DNA ("RNA-dependent DNA 
       polymerase")
   b. RNA is used as a template to direct 
       synthesis of new DNA at 3' end of 
       chromosome

Question 10

What do TRF1 and TRF2 do?

Answer 10

Proteins that bind to the 3’ end of one strand the 5’ of the other to protect the ends