Chen Yonggang

1
Biochemistry

• Chen Yonggang
• Zhejiang University
• Schools of Medicine

2
Central dogma

• replication
• transcription translation
• DNA RNA
  Protein

3
DNA Replication

• .

4
(No Transcript)
5
DNA Replication-Conservation of Information

• DNA replication must be carried out every time a
  cell divides
• Procaryotic growth involves cell division
• Mitosis in eucaryotes involves cell division
• DNA replication is template driven and
  synthesizes DNA in a semi-conservative manner
• dNTP DNAn DNAn1 PPi

6
DNA is replicated in a semi-conservative manner

• Messelson Stahl showed, using 15N-labelled DNA
  that the products of replication had intermediate
  density

McKee 18.2
7
Each separated DNA strand is duplicated to give
two new double helices.
8
DNA semiconservative replication

• Each DNA strand serves as a template for the
  synthesis of a new strand, producing two new DNA
  molecules , each with one new strand and one old
  strand, this is semiconservative replication.

9
The process which appeared simple initially is
complex

• Replication occurs just prior to cell division
• The E coli chromosome is a single circular DNA
  double helix associated with proteins in a
  nucleoid
• The E coli chromosome is negatively supercoiled
  and thus is quite compact and inaccessible

McKee 17.16
10
To allow the replication to occur supercoiling
must be relaxed

• A type I (single strand breaking) topoisomerase
  cleaves and relaxes the negative supercoiling
  ahead of the replication complex
• The topoisomerase has a central hole through
  which the double helix passes. An intermediate
  is an enzyme-linked 3OH
• dnaA is displaced providing access for the next
  component needed for replication

11
The first step in replication involves oriC

• OriC is a 245 bp region, the origin of E coli
  replication
• OriC contains 3 tandem repeats of a 13 bp
  sequence beginning in GATC and rich in AT bp
• These repeats are weakly H-bonded and serve to
  provide 4 binding sites for a protein, dnaA, a
  start signal for replication
• Replication proceeds in two directions -
  bidirectional

12
dnaA allows binding of two other important
proteins

• dnaB is a DNA helicase which carries out the ATP-
  driven unwinding of the DNA double helix
• dnaC is an important accessory protein which
  binds and is soon released
• Together the three proteins utilize ATP to bend
  and separate the two strands of the bacterial
  chromosome

13
SSB, a single strand binding tetramer stabilizes
the initiation complex
McKee 18.7
14
DNA replication involves many enzymes

• An RNA primase binds to the SSB stabilized melted
  helix
• Since DNA polymerases require a primer and only
  extend that primer, the RNA primase (dnaG) in
  association with other proteins (primosome)
  synthesizes a 5-7 nucleotide primer using
  information from the template strand

15
At the replication fork two strands are managed
differently

• The 5 end of the primer(leading strand) is
  extended continuously by DNA polymerase III in a
  5?3 direction by dNTPs
• The primer on the lagging strand is also extended
  5?3 by DNA polymerase III, in a discontinuous
  manner
• Thus primer is made once on the leading strand
  and every 1000 nucleotides on the lagging strand

16
The replication fork
McKee 18.6
17
DNA Polymerase III holoenzyme contains 10
distinct types of subunits

• DNA Polymerase III is the primary replicase in E.
  coli
• It has polymerase and 3?5 exonuclease
  activities
• It functions as a dimer
• It has great fidelity, only 1 error in 1010 bp
• It is highly processive, sticking to the DNA for
  the entire trip through the chromosome
• It has a rapid biosynthetic rate, synthesizing
  1000 nt/sec

18
The Pol III synthesizes DNA from dNTPs
McKee 18.3
19
The Dimer moves in one direction and synthesizes
5?3

• The leading strand is synthesized by addition of
  5-dNTPs in response to the template
• The looped lagging strand is synthesized in
  Okazaki fragments using 5-dNTPs
• The lagging strand must be pieced together using
  DNA Polymerase I

20
DNA polymerase III forms phosphodiester bonds

• 2-deoxynucleoside 5 triphosphates are the
  activated intermediates needed for synthesis
• Information from the parental strand provides the
  information for 5?3 synthesis (parental strand
  is read 3?5)
• Thus each parental strand serves as the template
  for synthesis of a complementary strand

21
Synthesis of a phosphodiester bond
22
DNA Polymerase III is at the center of Replication
McKee 18.8
23
Top-down view of replication
24
While DNA polymerase III does the replicating,
DNAP I cleans up

• Pol I(100kd) is a monomer of about 10 the size
  of Pol III(900kd)
• It has three activities
• It is a DNA polymerase
• It is a 3?5 exonuclease
• It is a 5?3 exonuclease
• It is a processing and proofreading enzyme

25
The three activities are on one polypeptide

• The larger fragment(Klenow fragment) of 67kd
  contains the polymerase and the 3?5 exonuclease
  activity
• The smaller, 36kd contains the 5?3 exonuclease
  activity

26
As Pol III finishes, Pol I goes to work

• The 5?3 exonuclease removes the RNA primer
• The polymerase synthesizes DNA to fill the gap
• Errors in Pol III synthesis are removed by 3?5
  exonuclease
• The function of DNA Pol II is not understood,
  although it apperas to be similar to Pol I

27
Supercoiling was taken out by dnaB, DNA gyrase
replaces it

• Following synthesis of the strands, excision of
  RNA, replacement by DNA using Pol I, the
  supercoiling can be reinstated
• DNA gyrase, an ATP- linked, energy requiring
  enzyme introduces negative supercoils to restore
  the original twist in the leading strand

28
DNA ligase seals the Okazaki fragments and the
completed double helical DNA

• DNA polyI removes the primers and fills the gaps,
  DNA ligase seals the nicks and Okazaki fragments
  are connected
• Pyrophosphate cleavage drives the reaction to
  completion
• Termination occurs at a ter region and is
  mediated by a binding protein TBP
• A type II(double stranded) topoisomerase is
  probably involved in helix dissociation(the two
  daughter DNA molecules separate)

29
Enzymes and proteins involved in DNA replication

• 1, Topoisomerase
• 2, dnaA 1 recognize the origin of
  replication
• dnaB(helixase)unwind double helix
• dnaC
  
• 3, SSB
• 4, Primase
• 5, DNA polymeraseIII, DNA polymeraseI
• 6, Ligase

30
Happy Birthday

• Samar and Barry

31
Eucaryotic Replication is similar to that of
procaryotes

• Both have initiation, elongation and termination
  phases and are bidirectional
• Both involve multiple DNA polymerases
• Both involve multiple copies of the primary
  replicase which replicates strands differently
• Replication rate is slower, but replication is
  rapid due to multiple replicons
• Both require topoisomerases to unwind and rewind
  the DNA
• Both require ligases

32
Eucaryotic replication is distinct from that of
procaryotes

• There are 5 polymerases(a,ß,?,d,e)
• The chromosomes are linear
• There are multiple ori, and replication units
• Replication only occurs during the S phase of the
  cell cycle
• Telomeres restrict the number of times a replicon
  can be expressed

33
Initiation of replication occurs at multiple ori

• A large complex of proteins assembles at an ori
  (Origin Recognition Complex-ORC)
• Details not for testing
• A complex with helicase activity must bind and be
  activated
• Replication Protein A (RPA ) binds and separates
  the strands(like SSB in E.coli)
• RFC(replication factor C a clamp loading factor)
  and PCNA(proliferating cell nuclear antigen)
  allows binding of Pol d to both the leading and
  lagging strand

34
Binding of initiation factors to the lagging
strand differs

• Pol d is the main eucaryotic replication
  polymerase (Details not for testing)
• Replication protein A(RPA) binds to the single
  strands
• Pol a and a primase complex binds to the lagging
  strand
• An RNA primer and 15-30 dNTPs are synthesized
• Pol d binds and replicates one nucleosomes worth
  of Okazaki fragment

35
Finishing and sealing of the lagging strand is
different

• Specific protein factors are important in
  finishing up replication (details not for
  testing)
• DNA polymerase ? remove the primers and DNA
  polymerase ? excise errors
• Topoisomerases induce supercoiling
• DNA ligase seals the breaks
• Chromosomes segregate
• Replication bubbles merge
• Telomeres determine the end of replication

36
Eukaryotic DNA polymerases

• Eukaryotes have at least 15 DNA Polymerases (5
  most important)
• Pol a acts as a primase (synthesizing a RNA
  primer), and then as a DNA Pol elongating that
  primer with DNA nucleotides. After a few hundred
  nucleotides elongation is taken over by Pol d and
  e.
• Pol ß is implicated in repairing DNA.
• Pol ? replicates mitochondrial DNA.
• Pol d is the main polymerase in eukaryotes, it
  is highly processive and has 3'?5' exonuclease
  activity.
• Pol e may substitute for Pol d in lagging strand
  synthesis, however the exact role is uncertain.

37
Telomeres are GC rich self-complementary
sequences at chromosome ends

• Telomerase maintains the telomeres
• Telomeres are repeat structures with a terminal
  loop
• At each replication the telomeres are modified
  using an integral RNA template
• Loss of telomeres limits replication
• Cancer cells lose control of their telomeres

38
Telomeres

• The structure at the ends of linear eukaryotic
• chromosomes, generally consist of many tandem
  copies of a short oligonucleotide
• sequence.

39
(No Transcript)
40
Supercoiled DNA in Prokaryote
41
Structure of Nucleosome
42
Negative and positive supercoil