leading strand
involved in bidirectional replication of DNA
it is synthesised continuously
lagging strand
synthesised discontinuously in a series of ozaki fragments
problem of replicating ends of telomeres
linear eukaryotic chromosomes
problem with lagging strand at new 5’ end because last RNA primer on lagging strand end is removed but cannot be replaced
telomerase
solves problem of replicating ends of telomeres
T enzyme adds new sequence near end which compensates for sequence loss at 5’ end of lagging strand
only active in germ cells (produce egg and sperm) and not in most somatic cells (body cells)
chromosomes are shrinking because they get shorter each time they are replicated
DNA polymerases of E. Coli
DNA pol I
DNA pol II
DNA pol III
DNA pol IV and V
DNA pol I
helps remove RNA primer and replaces with DNA in chromosome replication
has major role in repair of damaged DNA
polA gene
5’-3’ polymerase
3’-5’ exonuclease
5’-3’ exonuclease
16-20 nucleotides/ second polymerisation rate
DNA pol II
restarting replication when blocked by damaged DNA
has a role in DNA repair
DNA pol III
chromosome replication
much faster than DNA pol I which just synthesises short stretches of DNA
polC gene
5’-3’ polymerase
3’-5’ exonuclease
no 5’-3’ exonuclease
250-1000 nucleotides/ second polymerisation rate
DNA pol IV and V
allow replication to bypass some types of DNA damage
also involved in DNA repair
role of E coli DNA polymerases
synthesise DNA polymerase
have exonuclease activities- degrading nucleic acids from the end
3’-5’ exonuclease
degrades from 3’ end
involved in proofreading
- checks the nucleotide it has just inserted is correct
5’-3’ exonuclease
used to degrade the RNA primers at the end of Okazaki fragments
helicase
unwinds DNA duplex to produce replication fork
DNAB gene in e coli
single stranded DNA binding protein
SSB in E coli
keeps strands apart and helps prevent stem-loop formation
primosome
makes RNA primer
moves with lagging strand
contains products of DNAC and DNAG genes and others in E coli
DNA polymerase III in action
DnaB (helicase) unwinds the duplex
alpha subunits (catalytic core) synthesise DNA
Tau subunits ensure dimerisation of polymerase
beta claim encircles DNA
leading strand is made continuously by addition of nucleotides to the 3’ end as it passes through the catalytic core of the enzyme
when sufficient template DNA for lagging strand synthesis has unwound, primase synthesises an RNA primer for lagging strand synthesis
template strand is then pulled through the catalytic core allowing the addition of nucleotides to 3’ end of primer
conditions of dna POL III
DNA pol III is symmetrical, so the lagging strand template forms a loop, so it is pulled through in the same direction
replication protein A
coats strands, preventing them from binding back together or forming secondary structures
- does same job as aSSB
DNA pol epsilon
synthesises leading strand
FEN1
known as a ‘flap’ endonuclease’
cuts off the RNA primer and degrades internally rather than at the end as in DNA pol I
DNA ligase then fills the gap
during okazaki fragment synthesis
reaches the 5’ end of the next fragment
displaces the RNA primer used to initiate that fragment and ‘flaps’ around
supercoils
in cells DNA is supercoiled
can be useful for replication as it is more compact
can cause difficulties in replication
can occur in circular DNA molecules and in linear DNA molecules if they are constrained at the ends
positive supercoiling
when the right handed double helix conformation of DNA is twisted in a right handed fashion
DNA is overwound in front of the replication fork
negative supercoiling
twisted in a left handed fashion
- looser coiling, take a twist out
DNA is relatively unwound behind the replication fork
