| 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: 300-307
Outline:
A. DNA replication ... Basic Principles:
1. Overall Principles in One Sentence Statement:
DNA replication proceeds Sequentially and Semi-Discontinuously from one or more Unique, Heritable Origin(s) at one (UniDirectional) or two (BiDirectional) replication Fork(s) in an overall Semiconservative replication mode to a Terminus, followed by Daughter Chromosome separation and Segregation.
Three Processes or Stages in DNA replication:
1. Initiation: events
that occur at the Origin(s) to start replication
Temporal Control of Replication occurs at this stage: WHEN
to replicate
2. Elongation: DNA synthesis events that occur at the Replication Fork(s)
3. Termination: events
at the Terminus resulting in Separation of Daughter DNA Molecules
Replicon: a DNA molecule with sites needed for its self-duplication,
ie replication
Origin of replication is required ... and is ALL that is
required ...
Terminus is NOT required
2. Prokaryotes:
Single Origin of DNA replication usually ... unique, heritable, Consensus
Seq
Chromosomes are circular
Most replication occurs BiDirectionally, with some Plasmid
and Viral exceptions,
eg the ColE1 origin and its derivatives such as pUC, pBR322, etc
Get so-called Theta
Structures ... [Brown, Fig 12.6]
Autoradiogram for the E. coli chromosome ...
Terminus absent in most Plasmid and Viral chromosomes
BUT present on Bacterial chromosomes ... [Brown, Fig 12.22]
Evidence:
1. Autoradiography ...
Bidirectional Rep from multiple origins in cultured human cells
and from Drosophila embryo DNA
2. Electron microscopy ...
Theta structures
EcoRI digested ColE1 => Unidirectional Rep
Linear DNA: how to
replicate precisely the ends ??? ... [Brown, Fig 12.23]
Several solutions ...
1. begin PRECISELY at each end ... Protein mediated ... some viruses
and some plasmids
2. use a circular chromosome: no ends to worry about ... as above
3. use Telomeres, as is done in eukaryotic chromosomes
and YACs ... [Brown, Fig 12.24, 12.25]
4. have repeats at ends, with replication mode that involves "jumping"
or circularization, as is done in replication of the DNA intermediates of Retrovirus genomes ... [Brown,
Fig 13.34]
3. Eukaryotes:
Many origins of DNA
replication ... [Brown, Fig 12.6B]
Cloning of Autonomously Replicating Sequences - ARS elements ...
recall YACs
See also Journal Article 3 on eukaryotic origins of DNA
replication ...
Replication proceeds Bidirectionally
... [Brown, Fig 12.6B]
No well-defined Termini ... forks run into each other ...
Replication at Ends of Linear Chromosomes a unique problem: Telomeres
4. Elongation essentials:
a. Semiconservative Replication:
...[Brown, Fig 12.1, 12.2]
forks unwind, each DNA strand serves as template for DNA synthesis
Meselson-Stahl CsCl experiment: ... [Brown, Fig 12.3]
b. Semi-Discontinuous
DNA Synthesis at Forks: ... [Brown, Fig 12.10]
Comes from fact that ALL DNA polymerases synthesize DNA 5'
to 3'
Leading Strand is the Daughter DNA strand with 3'
end at fork
This strand is synthesized continuously
Lagging Strand is the Daughter DNA strand with 5'
end at fork
This strand is synthesized discontinuously as Short fragments
(Okazaki fragments) that are joined together
c. Sequential replication - Forks proceed along the DNA from Origin to Terminus
d. Topology problem
... How to unwind the DNA ???
Answer: Topoisomerases coupled with DNA Helicases,
e.g. DnaB Helicase
... [Brown, Fig 12.4, 12.5, 12.13, 12.14]
5. Initiation essentials: [Brown, Fig 12.6, 12.7]
a. Loop formed at Origin, Replication Fork proceeds in one direction
b. If this is all, then get Unidirectional replication
c. If forks proceeds in both
directions, get Bidrectional replication
This can occur via an Okazaki fragment on Lagging Strand from
First Replication Fork passing through the Origin and becoming
the Leading Strand from the Second Replication Fork ...
(with appropriate addition of second DNA polymerase system ...)
Thus, understanding the unique features of the Initiation process means just understanding the steps in getting the First Replication Fork going ...
B. Other Prokaryotic DNA
polymerases:
ALL known DNA polymerase
show the same mechanism:
5'->3' synthesis; requirements for Primer, Primer Terminus,
Template; dNTPs
E. coli has three DNA polymerases:
PolI, PolII, PolIII... [Brown, Table 12.2]
PolIII, together with other proteins comprising HoloPolIII,
is the Replicase
C. Mutants and a Genetic
Analysis of DNA replication:
Replication is an essential
process; therefore, need Conditional Lethal mutants,
eg Temperature Sensitive (ts) mutants:
grow at 30 C (permissive temperature), die at 42 C (restrictive
temperature)
DNA replication ts mutants: specifically blocked in DNA replication ... two types:
1. Quick stop or Immediate
Stop: cease DNA rep immediately at 42 C ...
Elongation mutants: dnaB, some dnaC, dnaX, dnaQ, dnaZ, dnaT, dnaG,
polC (dnaE), dnaN
2. Slow stop or Residual
Synthesis: complete rounds of replication, and then stop ...
Initiation mutants: dnaA, most dnaC, one dnaB
Mutants in the polA gene, encoding
PolI, are NOT conditional lethal
(except for one mutant, specifically in the 5'->3' Exo activity
...)
| 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