Outline

1
Outline

• More Mechanisms of Replication
• Termination
• Eukaryotic DNA Replication
• Mitochondrial DNA Replication
• Correction of mistakes

2
A Replisome
3

Figure 20.20
4
Pol III
Figure 20.20
Core
? complex
Pol III has a dimer of the core subunits, which
contain the polymerizing a subunits.
5
The fact that the Pol III core is a dimer
indicates that there is concurrent synthesis of
leading lagging strands by one holoenzyme.
b
b
b
b
SSB
Pol III Core
Pol III Holoenzyme minus beta clamp
6
? - Clamp exists free and as subunit of Pol III
holoenzyme


.
Donut-shaped Dimer.
Clamps a subunit onto DNA, and makes it highly
processive.
-
Fig. 21.15 in Weaver
7
b Clamp can slide off ends of linear DNA
Based on Fig. 21.13
8
Pol III core dimer synthesizing leading lagging
strands.
Tau subunit of Pol III binds to helicase.
9
DNA Replication the movie
10
b Clamp loader
( g 2 , d, d, c, psi)
g Complex of Pol III holoenzyme
- loads b subunit dimer onto primer
Order of events

• Uses ATP to open b dimer and position it at 3
  end of primer.
• Loaded b clamp then binds Pol III core (and
  releases from g).
• Processive DNA synthesis.

11

• Recycling phase
• Once Okazaki fragment completed, b clamp
  releases from core.
• b binds to g .
• g unloads b clamp from DNA.
• b clamp recycles to next primer.

12
Figure 21.28

13
Termination of DNA Replication
14
Terminating DNA synthesis in prokaryotes.
Each fork stops at the Ter regions, which are 22
bp, 3 copies, and bind the Tus protein.
Fig. 21.27
15
Decatenation of Daughter DNAs
catenane
Decatenation is performed by Topoisomerase IV in
E. coli. Topo IV is a Type II topoisomerase
breaks and rejoins 2 strands of a duplex DNA.
Fig. 21.28
16
DNA replication in Eukaryotes

• Eukaryotic DNA polymerases (5)
• - has primase activity
• d - elongates primers, highly processive, can do
  proofreading
• - DNA repair
• - DNA repair
• g - replication of Mitochondrial or Chloroplast
  DNA

17
Eukaryotic DNA polymerases do NOT have 5' to 3'
exonuclease activity. A separate enzyme, called
FEN-1, is the 5' to 3' exonuclease that removes
the RNA primers.


Eukaryotes also have equivalents to the Sliding
clamp PCNA (a.k.a. proliferating cell nuclear
antigen-3 copies bind DNA) SSB RP-A
18
Problem for eukaryotes Replicating the 5 end of
the lagging strand (because chromosomes are
linear molecules)
FEN1 removes primer
Gap generated by removal of the RNA primer
19
Euk. chromosomes end with many copies of a
special Telomeric sequence.
(3 copies on this chromosome end)
Cells can lose some copies of the telomere w/out
losing genes.
(Replication of this chromosome would produce 1
that is shorter by 1 telomere)
20
Telomere Sequences
Telomeres form an unusual secondary structure.
Dashes are Ts
5 3
21
Telomerase
Enzyme that adds new telomeric repeats to 3 ends
of linear chromosomes.
22
Figure 21.34
23
More on the importance of Telomerase

• Apoptosis - Cells are very sensitive to
  chromosome ends because they are highly
  recombinogenic. Telomeres dont trigger
  apoptosis.
• Aging - There are rapid aging diseases (e.g.,
  Werners Syndrome) where telomeres are shorter
  than normal.
• Cancer - Most somatic cells dont have
  telomerase, but tumor cells do. Over-expression
  of telomerase in a normal cell, however, wont
  turn it into a tumor cell.
• Plants - Transgenic Arabidopsis with the
  telomerase gene turned off developed normally up
  to a point, then became sick.

24
Mammalian Mitochondrial DNA (MtDNA)

• Multi-copy, circular molecule of 16,000 bp.
• 2. Encodes genes for respiration (13 proteins)
  and translation (22 tRNAs, 2 rRNAs).
• 3. 2 promoters (1 on each strand) the STOP
  codons for the protein genes, UAA, created
  post-transcriptionally by polyadenylation
• 4. Some genetic diseases caused by mutations in
  mtDNA. MtDNA mutations accumulate during aging.
• 5. MtDNA used to define phylogenetic
  relationships between species, subspecies, etc.,
  or define breeding populations.

25
Mt DNA replication
26
Mammalian (mouse) mtDNA Replication

• Two origins of replication H (for heavy strand)
  and L (for light strand) that are used
  sequentially for unidirectional replication.
• Persistent D-loop at H ori, which is extended to
  start replication of the H strand.
• Once 2/3 of H strand is replicated, L ori is
  exposed and replication of L strand starts.
• The lagging L strand replication gives 2 type of
  molecules a and b. b is gapped on L strand.
• b L strand finishes replicating, and then both a
  and b are converted to supercoiled forms.

27
Correction of mistakes in E. coli DNA
Polymerization
Error rate for DNA Polymerase 10-5 Error rate
for proof reading activity 10-5 Error rate for
E. coli DNA polymerization is 10-10-10-11
28
Marking newly synthesized DNA in E. coli
GATC normally methylated on the A CTAG

• Newly synthesized strands not methylated right
  away, delayed for 10 minutes gives
  hemi-methylated DNA
  
• GATC
• CTAG

Hemi-methylated DNA 1. Not recognized by the
oriC activation system 2. Recognized by the
Mismatch Repair System
29
Mismatch repair in E. coli
MutL and mutS proteins recognize mismatch, and
activate mutH. mutH nicks strand across from
nearest methylated GATC. A helicase
exonuclease degrade from nick to beyond the
mismatch. DNA Pol III ligase do repair
synthesis. Only for newly synthesized DNA.
Fig. 20.44
30
Mismatch Repair

• Repairs replication errors that create mismatches
• In E. coli, new DNA not methylated right away
• mismatch recognized by mutS, then mutL binds and
  attracts mutH (endonuclease that cleaves nearest
  CTAG that is not methylated)
• Eucaryotes have mutS and mutL homologues, but no
  mutH
• also have the requisite exonucleases, but not
  clear how the strand specificity is determined

31
Mismatch Repair and Colon Cancer

• Hereditary nonpolyposis colon cancer (HNPCC)
• 1/200 Americans is affected (15 of colon
  cancers)
• Characterized by microsatellite instability
• Microsatellites are tandem repeats of 1-4 bp
  sequences that change during lifetime of HNPCC
  patients
• Microsatellites are prone to replication slippage
  resulting in insertions or deletions, which are
  normally repaired by the Mismatch Repair (MMR)
  System
• Mutations in one of 5 mismatch repair (MMR)
  genes increase susceptibility to HNPCC

32
Correction of mistakes in E. coli DNA
Polymerization
Error rate for DNA Polymerase 10-5 Error rate
for proof reading activity 10-5 Error rate for
E. coli DNA polymerization is 10-10-10-11 Why
still 10-11? E. Coli genome is 4.2x106 One
mistake in every 2 rounds of DNA replication