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Lecture 15

BIO120H1 Lecture Notes - Lecture 15: Histidine, Only Time, X-Ray Crystallography


Department
Biology
Course Code
BIO120H1
Professor
Jennifer Harris
Lecture
15

Page:
of 6
Monday, September 15, 2008
Lecture 3: Intro to DNA Replication
Lecture Outline:
1) Overview of DNA replication
2) DNA replication in bacteria
Readings: Alberts textbook, Ch 5, pp. 263-276, 281-287
- Only time you need DNA replication is when cell divides.
- Every single cell in organism’s body contains entire genome required to build entire organism.
Questions of day
1) Is DNA replication conservative or semiconservative?
2) What is direction of DNA replication?
3) Where does DNA replication start?
- Watson & Crick worked out structure of DNA – used x-ray crystallography & models.
Is DNA replication conservative or semiconservative?
2 hypotheses:
1) DNA synthesis is conservative 2) DNA synthesis is semiconservative
Red strands? – Parental
Blue strands? – Newly synthesized
- Conservative 1 daughter cell (when cell divides) gets all
of original parental strand & other gets completely new
synthesized strand.
- Semiconservative use both strands as template, 1 of
original strand goes into 1 cell & other goes into other
cell. This hypothesis is right. Both daughter cells get 1 of original strand & 1 new synthesized strand.
- Important implications for error rates if you have 1 original copy & 1 newly synthesized copy, important
implications for how error checking may happen, how mistakes might be corrected, etc.
What is direction of DNA replication?
3 possible models:
1) Unidirectional growth of single strands from 2 starting points.
Ex: linear virus
2) Unidirectional growth of 2 strands from 1 starting point.
Ex: some plasmids
3) Bidirectional growth from 1 starting point. – This is most common type of replication.
Ex: eukaryotes
Where does DNA replication start?
2 possibilities:
1) Always start from same location on DNA have some control over process some consensus sequences that are
recognized as initiation for replication.
What are some of characteristics of sequences at replication origins?
-Easy to open, A-T rich 1st step in initiating DNA replication is to tear 2 strands of DNA apart b/c you
need to use 1 of them as template to synthesize new strand. A-T base pairs are weaker than G-C base pairs
– 2 H-bonds rather than 3.
- Attractive to initiator proteins bind to specific sequences proteins that help assemble machinery of
DNA replication at that site so that replication can begin.
2) Random start
How many origins of replication?
1) Single
Bacteria
2) Multiple Eukaryotes
- Ex: Yeast chromosome #3: 17 chromosomes & 400 origins interspersed at regular intervals generally
spaced at about 30 kilobases apart can be replicated in about 8 minutes. If you start deleting some of
these origins, replication machinery is fast enough that it can compensate for those losses but once you
get to certain # of losses, it actually overwhelms system & start to lose ends of chromosomes.
How do you identify origins of replication?
What does ARS stand for? Autonomously replicating sequences
- Plasmids found normally in bacteria can be inserted in any piece of
DNA.
- Purpose of experiment: what pieces of yeast DNA is the origin of
replication?
- Randomly chopped up random pieces of chromosomes & insert it into
thousands of plasmids. Take those plasmids & insert them into yeast cells 1
yeast cell gets 1 plasmid. Is there a sequence that will direct DNA to be
replicated? Origin of replication is named ARS. Grow them on medium – need
to find way to select yeast cells that contain origin of replication. Yeast with
its DNA machinery will make many copies of plasmid.
- Left: no replication of plasmids – no ARS – contains very few plasmids.
- Also include on plasmid gene called His gene allows bacterium (yeast) to
grow in medium without AA histidine.
- Take entire yeast genome & chop it up in little pieces put little pieces into plasmid vector. This plasmid
vector has gene which synthesizes histidine – significance of this ARS sequence is that it is term defined by
this assay as autonomously replicating sequence which means that if it contains this sequence this plasmid
will be able to replicate itself.
- If you have whole bunch of plasmids, each with slightly different portion of yeast genome, some of those
plasmids will have ARS sequences. If you introduce all of plasmids into yeast cells that actually lack this
His gene, that means that these yeast cells cannot actually grow in absence of histidine & then you played it
on selective medium without histidine, what you will find is that if that particular yeast colony has ARS
sequence in it, it will be able to replicate & therefore replicate His gene & therefore grow in medium
without His & then you can take these yeast colonies, extract plasmid from it, sequence piece of yeast
genome & determine what the sequence which is putative origin of replication might be is.
- ARS sequence is not entirely synonymous with origin of replication, it is putative origin of replication as
defined by this assay but in fact when you look at those sequences that result in ability to grow in medium
with His, many of them turn out to be origins of replication.
- In rare cases, plasmid without ARS sequence actually integrates into host genome & gets replicated there
very rare event but these colonies are result of that – false positives.
How does DNA replication proceed in bacteria?
This style of replication only applies to: circular genomes
- Have single origin of replication.
- When replication begins, it proceeds bidirectionally there are actually 2 replication
forks & eventually those 2 forks meet & you get dissociation & you have 2 complete
daughter DNA molecules.
- 1 of simplest systems.
What happens at DNA replication forks?
- In replication fork, there is 2 mechanisms/ways to replicate DNA:
leading strand (copy of parental) & lagging strand. Proceeds to right bit
choppy for lagging strand.
- Elongation of DNA strands in newly synthesized DNA always happens
in 5’ to 3’ direction. At replication fork, have leading strand (there is an
asymmetry that is happening here) that is able to be synthesized
continuously as replication fork moves in this direction & strands separate
whereas lagging strand is not able to synthesize DNA continuously got
to go in ratcheting fashion (backwards) has to be synthesized in short
chunks.
Overview of DNA replication
Procedure:
1) Separate DNA strands
2) Synthesize DNA
3) Proofread newly synthesized DNA (copy has to be exquisitely accurate)
Ingredients for synthesis:
1) Origin (certain sequence of DNA that initiator can recognize)
2) Primers (DNA polymerase can only elongate from existing primer sequence)
3) dNTPs (deoxyribonucleoside triphosphates – building blocks of nucleic acids)
4) ATP (for energy)
5) DNA polymerase (major enzyme that forms nucleic acid chain)
6) Accessory proteins
DNA synthesis
Base pairing: A-T, C-G
- Actual process of DNA synthesis is accomplished by polymerase. Incoming
nucleotide is determined by necessary base-pairing interaction with next AA in
template strand.
- Elongating chain is going from 5’ to 3’ – template strand is in opposite direction.
- Requirement is for DNA polymerase to keep elongating is that you have
correctly base-paired nucleotide at 3’ end if not, polymerase stalls & cannot
elongate further – part of error-checking mechanisms of cell.
Steps in bacterial DNA replication