Chapter 9 DNA replication
DNA replication; Meselson/Stahl experiments
Three possible modes of replication......Figure 9.1
The data (diagramatic): Figure 9.2
Predicted results for three models...Figure 9.1
Meselson-Stahl diagrams
to get generation 0
to get generation 1
to get generation 2
All together
note new book: Meselson, Stahl, and the Replication of
DNA: A History of 'The Most Beautiful Experiment in Biology'
by Frederic Lawrence Holmes
From: Meselson, M. and F. W. Stahl (1958). "The replication
of DNA in Escherichia coli." Proceedings of the National Academy of
Sciences 44: 671 - 682.
Meselson - Stahl experiments for isolated bacterial DNA; What about eukaryotic
chromosomes?
DNA replication; in chromosomes
First by J. H. Taylor using tritiated thymidine and autoradiography
also Incorporation of 5-bromodeoxyuridine (BUdR) into cells in culture
Secrets: BUdR (a base analog) replaces thymine in DNA replication;
if one of the two strands of the double helix have
BUdR the chromosome stains darkly but if both strands of
the double helix have BudR the chromosome stains lightly.
Harlequin chromosomes Figure 9.3 (not on web).. Harlequin.gif
Demonstration of semiconservative replication in DNA --Meselson and
Stahl
Demonstration of semiconservative replication in chromosomes -- Harlequin
chromosomes
Required materials for DNA replication
in vitro synthesis of DNA (in glass, in a test tube)
Konberg experiments..(page 188) DNA polymerase.. Kornberg.GIF
Importance of nucleoside triphosphate; what is a nucleoside?
Nucleoside - sugar and base with no phosphates;........ Nucleoside
triphosphate - sugar and base and three phosphates
5 November 2004
Review and add to DNA replication
Discuss properties of the telomere
Discuss the crossover model
Bidirectional replication of DNA in chromosome
A simplistic view first.
basic ideas
5' to 3' replication; need 3' end for DNA replication; RNA primer
inserted first to give 3' end (discussed in previous
lectures)
see DNA replication fork Fig 9.6
For eukaryotic chromosomes replication will begin at multiple
points, making for replication bubbles along the length of the chromosome.
Replication bubble..as in Figure 9.7..../bubble.GIF
simple animation DNA synthesis ...../DNArepl.html
Caution: when looking at one replication fork, we are looking at one
half of a bubble
Concept of replicon
"In eukaryotes the stretch of DNA from the origin of replication
to the two termini of replication (where adjacent
replication forks fuse) on each side of the origin is
called a replicon or a replication unit (Figure 9.9)"
--- text page 196
multiple points of origin of replication in eukaryotic chromosomes
(see Table 9.3); about 25,000 in the mouse
Why multiple origins? Speed of DNA polymerase 50 nucleotides
per second in a typical human cell. "At this
rate and with only one origin of replication, it would take
the polymerase about 800 hours, a little more than a month,
to copy the 130 million base pairs in an average human
chromosome." origins in humans given as 10,000 reference:
Hartwell, et al 2000. Genetics:From genes to genomes McGraw
Hill
Some enzymes at or in vicinity of replication fork
DNA helicase - break hydrogen bonds unwinds DNA at origin
primase - synthesis of RNA primer (about 11 nucleotides)
topisomerase - solve problem of super coiling
DNA polymerase-III - synthesis of DNA
DNA polymerase-I - removes primer and fills the gap
DNA ligase - unites okazaki fragments
Now back to telomeres
Observations of Barbara McClintock; loss of end of chromosome
(by radiation, breakage of paracentric inversion anaphase-I
bridge) leads to fusion of ends of chromatids
Telomere problem...3'overhanging end (ledge)
show end of chromosome T and D loops
Traced from cover of journal Cell volume 97(4) TelomereLoop.gif
and
from: Greider, C. W. 1999 Telomeres do d-loop-t-loop. Cell
97: 419-422..... TelomereLoop4.gif
from: Blackburn, E. H. (2001). "Switching and signaling at
the telomere." Cell 106(6): 661-73........ ../TelomereBlackburn.gif.
telomeric repeats
mammals - AGGGTT
Tetrahymena - GGGGTT
Arabidopsis - AGGGTTT
subtelomeric repeats, etc.
two problems with telomere
1. the single strand of DNA at the end of the chromosome.
Solution: D loop t loop
2. the inability to complete the replication of the end of
the chromosome. Solution: telomerase
the observation of Hayflick
The Hayflick Limit: limit to eukaryotic cell replications...Hayflick,
1961(cells in culture limit 40-60
replications)..Hayflick, L. and P. S. Moorhead.
1961 The serial cultivation of human diploid strains.
Experimental Cell Research 25: 585-621. (Leonard
Hayflick .... Susan)
Multiple solutions for loss of DNA - extension of 3' overhang by telomerase
also, unequal crossovers at the end of the chromosome
(Drosophila does not have telomerase, transposons at ends of chromosomes
- see Types of DNA handout
Events at the telomere are exceedingly complex. See Elizabeth
Blackburn's 2001 paper
Question about reduced telomere length in premature aging syndromes.
The answer is yes. See: Allsopp, R. C., et al. 1992 Telomere length
predicts replicative capacity of human fibroblasts. Proc Natl Acad
Sci U S A 89(21): 10114-8.
Also inherited disorder dyskeratosis congenita. See... Marciniak,
R. A., F. B. Johnson, and L. Guarente. 2000 Dyskeratosis
congenita, telomeres and human ageing. Trends Genet
16(5): 193-5.
8 November 2004
From the 28 October 2004 issue of Nature. Papers dealing with the
discovery of a new hominin species from an island in Indonesia. "Skeletal
remains show that the hominins, nicknamed 'hobbits' by some of their discoverers,
were only one metre tall, had a brain one-third the size of that of modern
humans and lived on an isolated island long after Homo sapiens had migrated
through the South Pacific region."
"'My jaw dropped to my knees,' said Perter Brown........"
for free papers, see:
To continue with the crossover discussion
The D-loop for both the tuck of the telomere and for the crossover is
really a displacement loop ... models show as a letter 'D'
Important items
the double strand break of one DNA molecule
the degredation of the 5' ends to form the gaps
The displacement of the 3' strand and the filling of the gaps with
DNA synthesis
this forms heteroduplexes, which may have nucleotide mis-matches.
there are two half DNA exchanges forming the Holliday junctions
the Holliday junctions are then corrected to form crossovers or no
crossovers
the mismatched base pairs may be corrected to form a gene conversion
the determination of gene conversion by using products of meiosis
which stay together
see ../OrderedMeioticProducts.gif
page 200 in the text has the original model Holliday for reciprocal
genetic recombination. It shows an electron microscope picture of the
plus shaped figure for two double helices. Figure 9.13 also shows hetroduplexes
and the correction of the Holliday junctions in either a horizontal cut
to the left giving no crossover and a vertical cut to the right giving
a crossover.
see Figure 9.13 in your text (not on the web)
... crossing
over
Holliday model - use rubber tube model for redraw
of Holliday intermediate
Electron micrograph of Holliday Junction (not on web here, see text
page 200) ... ../HollidayJunctionEM.gif
ref: http://gsbs.gs.uth.tmc.edu/courses/experimental_genetics/fig_7.jpg
