Major players at a DNA replication fork

Machines at the replication fork

Polymerases at the
replication fork

• semiconservative
• discontinuous
• replication fork geometry
• newly synthesized leading &
lagging strands
• all enzymes
• nucleic acid polarity
1
• all nucleic acids are synthesized in the 5’->3’ direction
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 Prokaryotic and eukaryotic DNA replication forks
have similar replication machines

Leman and Noguchi (2013) Genes 4: 1-32.

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 Is there is something special about chromosome ends?

telomeres

……TTAGGG - 3’
……AATCCC - 5’

3
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 Linear chromosomes: end replication problem at the telomere

4
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 The telomere length declines with age.

human granulocytes and lymphocytes from peripheral blood

5
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 Human telomere sequence

……TTAGGG - 3’
……AATCCC - 5’

6-bp tandem repeats X ~2000 copies/telomere = ~12,000 bp (wide range)

mouse = ~35,000 bp (wide range)

DNA replication leads to telomere erosion: ~200 bp lost/replication

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 <- cell doublings

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 8
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 9
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 Tetrahymena thermophila (ciliate)

Micronucleus – (germline, not expressed…)

Macronucleus – same genome as micronucleus,

but highly expressed. Chromosomes amplified
~45 X, then cut into smaller pieces, all of which
have two telomeres… 10
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 Looking for an organism with a lot of telomeres

Species Nucleus Mb (1n) chr (1n) telomeres

Human 1 3.1 Gb 23 92 (2n)

Tetrahymena MIC 104 Mb 5 20 (2n)

MAC 104 Mb 225 ~10,000 (~45n!)
corrected

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 Telomerase - reverse transcriptase [1]

Subunit Gene Size

Telomerase RNA component TERC 451 nuc [2]
Telomerase-associated protein TP1 2627 aa
Telomerase reverse transcriptase TERT 1132 aa

[1] 5’ -> 3’ RNA-dependent DNA polymerase

[2] includes the sequence 5’-CAACCCCAA-3’

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 Tetrahymena telomeres

5’ – TTGGGG – 3’
3’ – AACCCC – 5’

Sequence of a hypothetical Tetrahymena telomere (not to scale) ~50 repeats

3’ AACCC 5’
5’ TTGGGGTTGGGGTTGGGGTTGGGG 3’

So how does telomerase maintain chromosome length…?

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 Telomere lengthening: step 1

RNA template is 9 bases long, whereas

telomere DNA repeat is 6 bases long,
which allows telomerase to conduct
repeated rounds of translocation and
elongation…

Black letters – existing telomere DNA

Red letters – template sequence in TERC RNA
Blue letters – newly synthesized DNA

Tetrahymena sequence
……TTGGGG - 3’
……AACCCC - 5’

Human sequence
……TTAGGG - 3’
……AATCCC - 5’

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 Telomere lengthening: step 2 FILL IN THE GAP

Once the template (lower)

strand is extended sufficiently
by telomerase, DNA primase
can make an RNA primer, and
DNA polymerase can fill in the
“gap”, lengthening the
telomere… still retain 3’
overhang, but the chromosome
has been lengthened.

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 Shelterin shapes & protects telomeres

Shelterin: heterohexamer of
6 different proteins

t-loop shelterin

100-200 nucleotides

Protection of Telomeres (POT1)

16
Genes Dev.19: 2100-2110 (2005) Nature Reviews Cancer 8: 450-458 (2008)
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 Telomere loops: 3’-overhang strand invasion to form D-loop
Post class addition

5’
AGGGTTAGGGT
T T
T A
5’---TTAGGG AGGGTTAGGG3’ GGGTTAGGG---------
3’---AATCCCAATCCCAATCCCAATCCCAATCCC---------

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