DNA Ligase

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DNA Ligase

• Energy-dependent joining of the chains
• Activated by NAD or ATP hydrolysis NAD ? NMN
  AMP ATP ? AMP PPi
• AMP -attaches to lysine group on enzyme
• AMP transferred to 5 phosphate at ligation
  site
• 3 OH at ligation site splits out AMP and joins
  to 5 phosphate

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Ligase Mechanism
NAD
Activated Phosphorylating complex
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(No Transcript)
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AMP
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Eukaryotic Chromosomes have Telomeres
Rule The lagging strand at the extreme 5 end
of linear chromosomes cannot be accessed by DNA
polymerase
Solution Devise a method to extend the 5end
consisting basically of non-sensible DNA, to
extend the end
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Telomerase Action
1. Enzyme binds to TTG
2. Using enzymes RNA template and polymerase,
extends 3end of lagging strand
RNA
3. Shifts position to increase length of lagging
strand
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The Legacy of Telomeres

• Gradual loss foretells cell death
• Maintenance signals cell immortality
• Most normal cells have no telomerase
• Cancer cells have telomerase
• Progeria, premature aging, associated with low
  telomerase activity
• Aging in general may be telomere-related

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Reverse Transcriptase
RNA retroviruses eg., HIV, breast cancer
Used to make DNA from an RNA template
Major cloning tool
Product is called a cDNA (complimentary DNA)
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DNA REPAIR

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Why the Need for DNA Repair

• Chemical modification
• Alterations in the H-bond donor-acceptor pattern
• UV damaged DNA and thymidine dimers
• Methylation and alkylation of DNA
• Point mutations Transitions and
  Transversions
• Insertion/deletion mutations

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Types of Damage
Oxidative
NH4
O2
Cytosine
Uracil
Corrected by removing U
If not corrected
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Oxidative deaminations can occur from nitrous
acid, derived from sodium nitrite, used as a
food preservative
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UV Radiation
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Methylation
Only one in vertebrates
Methyltransferases protect bacterial DNA from
their restriction nucleases
Methylation distinguishes parental strand from
daughter strand
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Rule 80 of human cancers are caused by
carcinogens that damage DNA or interfere with
replication or repair
How can one spot a mutagen?
1. Animal Studies
Long and inconclusive
2. Ames Test
Histidine deficient Salmonella typhimurium (his-)
Add suspected mutagen
Add liver extract
Test for spontaneous revertants to (his)
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Two Ways to Maintain a Stable DNA
A replication process of high accuracy
Proofreading by 3- 5 exonucleases
One error for every 108-1010 bases incorporated
Correcting Genetic information when DNA is
damaged or modified chemically
Chemical modification of nucleotides
Photochemical changes
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Correcting Post-Replication Errors in DNA
or
DNA Repair
Rule The repair of DNA is a continuous ongoing
event that is linked to a cell surviving free of
mutagenic alterations
FACT An estimated 10,000 bases are set free in
DNA every day through breakage of
glycosidic bonds
Rule There are many, many ways to repair damage
to DNA
Rule Repair mechanisms tend to overlap and vary
in their efficiency and effectiveness of repair
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Examples of redundancy Repair of thymidine
dimers
1. Photolysis enzyme Reduction and bond
splitting
2. UvrABC endonuclease nucleotide excision
Why Overlap?
1. Because photolysis enzyme is designed to
spot the dimer through interaction with active
site on the enzyme.
2. UvrABC is designed to recognize alterations
in the helix structure and thus excises the bases
in the area around any nucleotides that distort
the DNA
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Types of DNA Repair
1. Direct Repair Intact Repair (no
phosphodiester bonds are broken during the
repair)
Examples
O6-methylguanine-DNA methyltransferase
inactive
active
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Excision Repair Enzymes

• UvrABC endonuclease (helix distortions)
• DNA glycosylase (damaged base)
• AP endonuclease (missing base)
• Uracil N-glycosylase (uracil in DNA)

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Excision Repair
UvrABC endonuclease
DNA glycosylase
Pol I
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Double Strand Breaks - Recombination - Error Prone
Rule When both strands of DNA are damaged,
excision repair has no means to gauge a repair
Rule Interruptions in the movement of the
replicating fork elicit a higher order repair
system called SOS
Rule Recombinatorial DNA repair or
error-prone repair is activated whenever DNA
damage occurs at a high level.this is the SOS
response
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SOS and Recombination Repair

• Occurs when damaged DNA is being replicated
• Controlled by RecA and LexA
• Error prone
• No template to guide
• Operates by genetic recombination via RecA

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SSDNA-RecA
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Recombinatorial Repair
Error-prone Repair
SOS
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Mechanism of RecA in Recombination Repair
1. Formation of RecA filaments
2. Alignment with homologous dsDNA
3. Unraveling and binding the replacing strand
4. ATP-dependent repair
5. Displaced intact ssDNA template