| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
Readings: Brown, 12: 307-318
Outline:
A. The Elongation Process in
Prokaryotes
... [Brown, Section 12.3.2]
HoloPolIII is essentially a Dimer Complex, but has Asymmetry due to Gamma-Delta-Delta' complex required to form a Beta Clamp for each new Okazaki fragment ...
This Asymmetry is important in dealing with a basic problem at Rep Forks:
1. How can DNA be synthesized at the 5' end of one Daughter Strand?
Replication proceeds SemiConservatively:
... [Brown, Fig 12.1, 12.2]
each Parental Strand serves as a Template strand, the growing
Daughter Strands serve as Primers ... ok for one Daughter Strand,
with 3'OH end, the Leading Strand ...
but not for the other, with 5' end, the Lagging Strand
Solution: ... [Brown, Fig
12.10]
The Lagging Strand is synthesized as Short Fragments in the
opposite direction to that of Fork movement.
These "Okazaki" Fragments are about 2000 bases in bacteria
and about 400-500 bases in eukaryotic cells.
2. Second Problem: What
is the Primer used in synthesis of Okazaki frags?
Solution: Short transient
RNA molecules, 1-15 bases long
... [Brown, Fig 12.10]
B. Proteins involved in Prokaryotic
DNA Replication:
Purify such proteins
either by:
1) Complementation of
dna mutants:
e.g. purify DnaB helicase via complementation of an E. coli dnaB
extract for in vitro replication
or by:
2) Reconstitution ... add fractionated extracts together to regain in vitro replication activity
Assay system (in vitro replication
system): small E. coli
phage phiX174, G4, M13 ...
used as "probes" of E. coli replication enzymatic apparatus
(Note: there is a good introductory discussion of these
small phage systems in Voet-Voet...)
Found: large number of Proteins needed for Elongation at Fork
in E. coli
Several of these form a complex with PolIII:
1. HoloPolIII Replicase
Subcomplexes: about 20 polypeptide chains involved ... [Brown, Fig 12.16]
1. Catalytic Core: PolIII
... Alpha subunit ... polC = dnaE gene ... polymerization
Epsilon and Theta subunits ...
Epsilon: dnaQ gene ... 3'->5' Exo: proofreading ...
[Brown, Fig 13.3]
2. PolIII' = Core + Tau:
2x Core + 2x Tau subunit: dimer enzyme ... increased
processivity
One half for the Leading Strand, other half for the Lagging Strand
Tau and/or Theta are thought to function by associating
the two PolIII Core complexes together, linking Leading and Lagging
strand synthesis together
3. Gamma-Delta-Delta'-Chi-Psi
complex
This Gamma-Delta complex is thought to be the ATPase that loads
the Beta Clamp: Clamp Loader
Gamma and Tau both come from dnaX gene, different transcripts
4. Beta Ring or Clamp:
dnaN gene product ... 2 homo dimers, bind both template strands
at forks
Two Beta subunits form a ring around the DNA
This Beta Ring directs formation of the rest of the HoloPolIII
proteins on the DNA
and serves as a Clamp to hold HoloPolIII on the DNA ...
increased processivity
The Beta Clamp is present
on both Leading and Lagging strands, forms once on the Leading
Strand at the beginning of Elongation but forms on the Lagging
Strand with each Okazaki Fragment ...
This occurs in an ATP-dependent reaction, requiring the Gamma-Delta-Delta'-Chi-Psi
complex
5. Gamma-Delta-Delta'-Chi-Psi
complex on Lagging Strand only ...
Loading of Beta Clamp is required for each Okazaki fragment,
and hence the Gamma-Delta complex is needed continuously for the
Lagging Strand ...
but loading of Beta Clamp occurs only once on the Leading Strand,
at time of initiation ...
Hence HoloPolIII is thought to be assymetric, with one half (the
half for Lagging Strand synthesis) having the Gamma-Delta complex,
but the other half (for Leading Strand synthesis) lacking this
Gamma-Delta complex.
2. Other Proteins which catalyze these and other events in Elongation:
1. Helicase and Topoisomerase
activities: open up the Parental Strands
Rep protein: 3'->5' helicase ... essential in phiX174 replication,
not essential in E.coli
DnaB Helicase: 5'->3' helicase ...most important
in E. coli ...
[Brown, Fig 12.4, 12.5, 12.13, 12.14]
2. SSB protein: Single-Strand Binding protein ... stabilizes exposed Parental strands
3. Primosome:
Complex of DnaC, DnaB, DnaT proteins with Priming Factors
Primosomes assemble at Primosome Assembly Sites (PAS): specific
DNA sites
Once assembled, the Primosome can move in 5'->3' direction
to site of RNA syn
DnaG protein, a Primase, binds to the Primosome and synthesizes the short RNA piece that serves as a Primer for Okazaki fragment synthesis ... [Brown, Fig 12.12]
DnaB protein directs Primosome to RNA syn site,
facilitates Primase binding
SSB protein is displaced by Primosome in this movement
4. HoloPolIII displaces Primase, uses the RNA as a Primer, and synthesizes O.frags
5. When the head of one O.frag hits the RNA tail of an adjacent O.frag, HoloPolIII ceases DNA synthesis.
6. DNA polymerase I
takes over: Nick Translation through the RNA primer
5'->3' Exo activity removes the RNA primer, while simultaneously
synthesizing DNA in this Lagging Strand fragment where the RNA
was. ... [Brown, Fig 12.17]
7. DNA ligase then joins the Okazaki fragments together by sealing the Nick
8. DNA gyrase introduces
appropriate negative SuperCoils
C. Eukaryotic DNA polymerases
and Replication Proteins:
5 types of mammalian
DNA polymerases, with cognates in lower eukaryotes:
... [Brown, Table 12.2]
1. DNA polymerase Alpha
: Lagging strand synthesis
has both Primase and Polymerization activities
2. DNA polymerase Delta
: Leading strand synthesis
Thus, have separate Polymerases, but joint Primase-Polymerase
3. DNA polymerase Beta and Epsilon : DNA repair enzymes
4. DNA polymerase Gamma
: Mitochondrial DNA replication
Other Replication Proteins and Sites:
1. ARS - Autonomously Replicating
Sequences
these sequences contain the DNA sequences used for initiation:
Eukaryotic Origins
examination of many such ARS sequences gives a Consensus
Sequence: [Brown, Fig 12.8]
ARS sequences from one eukaryotic species, e.g. humans, work as
origins in other eukaryotic species, e.g. yeast ... this is true
ONLY for prokaryotic origins of DNA replication from "closely"
related bacteria ... see discussion below on Eukaryotic Replication Origins from Journal Article 3
1. PCNA (Proliferating
Cell Nuclear Antigen): ... [Brown, Page 311]
coordinates Leading and Lagging Strand synthesis events
2. Replication Factors:
SSB ... Helicase activities ...
3. Topoisomerases I and II
4. RNaseH and Okazaki Fragment
maturation ... [Brown, Fig 12.19]
D. Initiation of Rounds of DNA Replication at E. coli oriC
1. Properties of oriC: ... [Brown, Fig 12.7A]
4 direct and inverted repeats of the 9-bp sequence TTATCCACA
3 direct repeats of a 13-bp sequence that begins with GATC
10 copies of the 4-bp sequence GATC
Positions and sequence of these sites is nearly perfectly conserved between 6 bacterial origins from Enterobacteria (E. coli, S. typhimurium, K. pneumoniaea, etc), and all of the origins function as origins in E. coli, using E. coli initiation proteins ...
These elements and their spacing
then define the Enterobacterial Origin of DNA rep
2. Protein Complexes formed in Initiation in E. coli: ...
[Brown, Fig 12.7B]
1. Initial complex: DnaA protein, interacting with 9-mers ... 20-40 DnaA molecules
2. Open complex: DnaA interacts with 13-mer direct repeats, opening up oriC in region of the 13-mers
3. Pre-Priming complex: DnaB-DnaC hexamers bind, form one or both Rep forks
4. Remaining events are analogous
to those during Elongation at Rep forks:
Primosome is formed: Primase (DnaG), DnaB -PAS ... RNA
for O.frag made
HoloPolIII binds to replicate both Leading and Lagging Strands
PolI and Ligase maturate and join O.frags
Gyrase introduces supercoils ...
E. Initiation of DNA Replication in Eukaryotes
1. Proteins - SV40 ... T antigen mainly ... Fig 2, J. Art 3
2. Proteins - Yeast paradigm - Fig. 1, DePamphilis J. Article:
J Art 3
a. ORC (Origin Replication Complex) proteins (6 in yeast)
bind after Mitosis
b. Cdc6 protein binds
c. Mcm (MiniChromosome Maintenance) proteins (6 in yeast)
bind in Early G1
This yields the Pre-Replication Complex
d. kinase activity replaces Cdc6 with Cdc45 in yeast ... Origin
Decision Point
This occurs in Late G1 ... creates a Pre-Initiation Complex
e. activation of Pre-Initiation Complex in S phase to initiate
rounds of DNA rep.
3. Eukaryotic Replication
Origins:
a. Simple ... SV40
... Fig 2, J. Art 3
b. Yeast ARS: ... Fig 2, J. Art 3; [Brown, Fig 12.8]
A, B1 regions for ORC binding;
B2 as DUE (DNA Unwinding Element - AT rich region);
B3 as Transcription Factor binding site
c. S. pombe: ... model for higher eukaryotes ... Fig. 2, J.
Article 3
Initiation Zone (5 - 50 kb) containing three ARS sites
(~200 bp)
d. Mammals: ... Fig 2. J. Article 3
Initiation Zone with ARS; role of other factors important ...
Need at least an ORC binding site plus a DUE
4. Other Factors determining
if a Potential Origin is Used
OBR - Origin of Bidirectional Replication ... DePamphilus, J. Article 3, Fig 3
actual site at which bidirectional replication is initiated ...
a. Nuclear Structure: see DePamphilus J. Article 3
1) Nucleus serves to concentrate needed protein factors
2) Newly synthesized DNA is associated with 'Nuclear Matrix'
in nucleus
3) Nuclear Structure may be used to establish sites for initiation.
This is correlated with the Origin Decision Point in Late
G2
and with a need for an intact Nucleus
b. Chromatin Structure:
1) can either enhance or inhibit origin activity via access to
transcription factors
2) Histone H1 decreases frequency of initiation => solenoid
structure not good
3) Presumably, heterochromatin would inhibit origin activity
c. Transcription: ... can either enhance or inhibit origin
activity
1) Transcription factors can enhance origin activity via their
binding
2) Transcription through a potential origin can inhibit it being
used
d. DNA methylation: ... methylation of C's in CpG islands:
-CGCGCGCG-
1) Some Initiation Zones have CpG islands ... and these are heavily
methylated
2) Other Initiation Zones have no CpG islands ...
Thus may be important in some Origins but not in others
...
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith