replication DNA Part 4
http://blogs.jobdig.com/wwds/les/2008/08/lobsters_500.jpg
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Last Time
The gamma complex of DNAPIII and the role of
the beta clamp
Ring on a String experiments that demonstrate
how the B-Clamp associates with DNA
Stewart, J. et al. (2001) results show how the
gamma complex associates with the B-clamp.
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Learning Outcomes
By the end of this lecture, you should be able to:
1. Describe the process of replication initiation and
termination (in proks).
2. Describe how the origin of replication was identied
(experiment) and be able to apply this technique to a
new problem.
3. Describe mtDNA replication and identify ways it is
both similar to and different from E. coli replication.
4. Illustrate how telomerase works.
5. Discuss how mtDNA mutations and telomere
mutations are associated with aging phenotypes.
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DNA replication Pt 4
1. Three phases of prokaryotic DNA
replication (experiment: dening the origin of
replication)
2. Replication of mtDNA
3. Replication in Eukaryotes
Image courtesy: http://www.brunel.ac.uk/2206/SHSSC/Telomerase-2.jpg
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Three phases of DNA replication
1. Initiation
2. Elongation
3. Termination
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Initiation
How is an origin of replication selected?
Primosome assembly at the origin
The oriC (Bacterial) Origin
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Experimental Question: what portion of
DNA is crucial for replication to occur?
purify Amp gene
Fig 21.3
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reiterative
Fig 21.3
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OriC
consensus
sequence
245 bp minimal region
required for replication
initiation in E.coli
Comparison with other
bacterial genomes
shows this region is
highly conserved!
Fig. 21.4
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Fig. 21.4
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DnaA boxes:
Fig. 21.4
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+ ATP
+ Hu on the origin
Ready to bind primase (DnaG)!
Similar to Fig. 21.5
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Interaction of DnaB,
DnaG and Pol III
DnaB helicase
DnaG
primase
leading strand
lagging strand
From: Lewin, Genes (2000)
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Initiation Summary
1. Replication begins at a ~245bp origin of replication, a highly
conserved region called oriC in bacteria.
2. Many copies of dnaA protein bind the four 9-mers within oriC;
DNA wraps around dnaA forming Initial Complex.
3. This triggers the opening of the AT rich 13-mers (Open Complex).
4. Two copies of helicase (dnaB) bind the 13-mers.
5. Primase (dnaG) binds to helicase (dnaB) and the DNA.
6. Helicase:primase complex moves along the template 3-5
synthesizing RNA primers 5-3 for DNAPIII to extend.
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DNA replication Pt 4
1. Three phases of prokaryotic DNA
replication (experiment: dening the origin of
replication)
2. Replication of mtDNA
3. Replication in Eukaryotes
Image courtesy: http://www.brunel.ac.uk/2206/SHSSC/Telomerase-2.jpg
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Mitochondrial DNA (mtDNA)
replication
http://img.webring.com/r/m/mito/logo
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Mitochondrial DNA (mtDNA)
replication
Multi-copy, circular molecule of ~16,000 bp
Codes for respiration proteins (13 proteins) and
translation (22 tRNAs, 2 rRNAs)
Some versions of genetic diseases caused by
mutations in mtDNA (Parkinsons, Leighs)
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mtDNA mutations accumulate during aging
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http://upload.wikimedia.org/wikipedia/commons/thumb/d/dc/Mitochondrial_DNA_and_diseases.png/
724px-Mitochondrial_DNA_and_diseases.png
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mtDNA replication
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http://dir.niehs.nih.gov/dirlmg/images/Mito.jpg
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mtDNA replication
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Fig 15.16 Lewin, Genes IX
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mtDNA replication
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Fig 15.16 Lewin, Genes IX
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Fig 15.16 Lewin, Genes IX
Completion of mtDNA replication
Nicks
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Mitochondrial DNA (mtDNA)
replication summary
1. Two origins of replication: H (heavy strand) and L (light
strand), used sequentially for unidirectional replication
2. Persistent D-loop at H ori, which is extended to start
replication of the H strand
3. Once ~2/3 of H strand is replicated, L ori is exposed and
replication of L strand starts
4. The lagging L strand replication results in 2 types of
molecules:
and . is gapped on new H strand.
5. L strand nishes replicating, and then both and are
converted to super-coiled forms.
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DNA replication Pt 4
1. Three phases of prokaryotic DNA
replication (experiment: dening the origin of
replication)
2. Replication of mtDNA
3. Replication in Eukaryotes
Image courtesy: http://www.brunel.ac.uk/2206/SHSSC/Telomerase-2.jpg
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There are ve eukaryotic DNA
polymerases
- has primase activity (makes an RNA primer, but
elongates using DNA nts!!)
- elongates primers on lagging strand
- elongates primers on leading strand (in yeast 2007*)
- DNA repair
- replicates Mitochondrial or Chloroplast DNA
*Science (2007):317(5834):127-130
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Eukaryotic polymerases DNA do
not have 5-3 exonuclease activity
FEN-1 is the 5' to 3' exonuclease that removes
the RNA primers (like DNAPI in prokaryotes)
Other Eukaryotic equivalents:
clamp in Proks = PCNA in Euks(a.k.a.
proliferating cell nuclear antigen)
SSB in Proks = RP-A (human/Euks)
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3
5
3
5
5
3
5
3
5
3
3
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3
5
3
5
5
3
FEN1
5
3
5
3
3
5
3
5
3
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3
5
3
5
5
3
FEN1
5
3
5
3
3
Gap generated by
removal of the RNA primer
5
3
5
3
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In most cells, chromosomes become
shorter with each replication cycle
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In cells in which telomerase is active, DNA
sequence at ends is recovered
Image courtesy: http://www.brunel.ac.uk/2206/SHSSC/Telomerase-2.jpg
Regions of active telomerase
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How telomerase works
Fig 21.34
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How telomerase works
(continued):
Fig 21.34
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note that there will always be an
overhang after removal of primer!
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Why is this a problem?
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Tying up loose (telomeric) ends: The T-loop model
http://www.mun.ca/biochem/courses/3107/Lectures/Topics/Euk_replication.html
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B R I E F C O M M U N I C AT I O N S
Common variants near TERC are
associated with mean telomere length
Veryan Codd1,11, Massimo Mangino2,11, Pim van der Harst1,3,11,
Peter S Braund1, Michael Kaiser1, Alan J Beveridge1, Suzanne Rafelt1,
Jasbir Moore1, Chris Nelson1, Nicole Soranzo2,4, Guangju Zhai2,
Ana M Valdes2, Hannah Blackburn4, Irene Mateo Leach3,
Rudolf A de Boer3, Masayuki Kimura5, Abraham Aviv5,
Wellcome Trust Case Control Consortium10, Alison H Goodall1,
Willem Ouwehand6, Dirk J van Veldhuisen3, Wiek H van Gilst3,
Gerjan Navis7, Paul R Burton8, Martin D Tobin8, Alistair S Hall9,
John R Thompson8, Tim Spector2,11 & Nilesh J Samani1,11
We conducted genome-wide association analyses of mean
leukocyte telomere length in 2,917 individuals, with follow-up
replication in 9,492 individuals. We identified an association
with telomere length on 3q26 (rs12696304, combined
P = 3.72 1014) at a locus that includes TERC, which encodes
the telomerase RNA component. Each copy of the minor allele
of rs12696304 was associated with an ~75-base-pair reduction
Wednesday,telomere length,2011
in mean September 7, equivalent to ~3.6 years of
to human chromosomes 3p26.1, 10q26.13, 12q12.22 and 14q23.2
(refs. 35). A recent genome-wide association study (GWAS) identified associations of two SNPs on chromosome 18q12.2 with telomere length, although the associations were not at a genome-wide
significant level6. To identify additional variants that affect telomere
length, we undertook genome-wide association analyses in two large
European cohorts followed by replication of promising signals in
three further European cohorts.
The discovery cohorts comprised 1,487 individuals with coronary
artery disease from the British Heart Foundation Family Heart Study
(BHF-FHS)7 and 1,430 United Kingdom Blood Service donors (UKBS)8
for whom there was genome-wide SNP genotype data available that was
generated using the Affymetrix 500K array as part of the Wellcome
Trust Case-Control Consortium (WTCCC) study8. Further details of
the cohorts and genotyping procedures are given in the Supplementary
Methods and Supplementary Table 1. Mean leukocyte telomere length
was measured using a quantitative PCRbased technique9 that expresses
mean telomere length as a ratio (T/S) of telomere repeat length (T) to
the copy number (S) of a single-copy gene, 36B4, within each sample
(Supplementary Methods). The T/S ratios were distributed normally
(Supplementary Fig. 1a) and showed the expected age-related attrition
in telomere length (Supplementary Fig. 1b) in both cohorts.
Nat Gen (2010) 42: 197
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DNA replication
Summary
1. Three phases of DNA replication: Initiation,
Elongation, Termination
2. Replication in Eukaryotes: Five polymerases, some
functionally similar replication proteins in proks
and euks
3. Special issues in Eukaryotic DNA: Some cells
maintain 5 ends after replication with Telomerase
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Next time:
Chromatin
structure and
gene
expression
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Additional questions
1. Why are telomeres necessary in eukaryotic replication?
2. What is the role of TERC?
3. How is mtDNA replication different than genomic DNA
replication?
4. If you were to perform the mustache experiment in
mitochondria, what would you expect to see?
5. Dene D loop, L strand and H strand
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de Lange T Genes Dev. 2005;19:2100-2110
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Some Review Questions
1. Catenane structure can be resolved using which enzyme?
a. Topoisomerase I
b. DNA gyrase
c. Topoisomerase IV
d. Type I topoisomerase
2. Which of the following are found in the Initial Complex at the
replication origin in E. coli
a. DNA polymerase
b. DnaA
c. DnaB
d. SSB
3. True of False. Primase is the first enzyme to attach to the
newly melted DNA strands at the OriC.
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