Wednesday, May 9

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Wednesday, May 9 400  PM         Professor Ivan
Dmochowski-University of Pennsylvania         "Che
mical Tools for Biomolecular Imaging"
        PSB 4606
Thirty-Third Annual B.R. Baker LectureThursday,
May 10, 2007400 PM ESB 1001Professor Vern
SchrammYeshiva UniversityAlbert Einstein
College of Medicine"Enzymatic Transition
States, Analogues and Applications"
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Wednesday May 16, 2007300pmPsych 1802BRUCE
LITTLEFIELDChanging Paradigms in Cancer Drug
Discovery Targeted Thearpies Natural Products,
and Unexpected Opportunities
Thursday, May 17, 2007330pm Rathmann
Auditorium (LSB 1001)VIRGINIA MILLEREvolutional
snapshot of the pathogenic Yersiniae
Friday, May 18, 2007300pmWebb Hall 1100BILL
GOLDMANPhase-specific Genes and Virulence of
Histoplasma capsulatun
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Chapter 25 DNA Metabolism
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• DNA metabolism.
• DNA replication needs to have high fidelitybut
  not too high
• Fidelity determined by basepairing, polymerases
  and repair
• DNA is dynamic.genetic rearrangements

Terminology dna, rec genes
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25.1 DNA replication Semiconservative Origins Rep
lication forks 5?3 direction Nucleases and
polymerases Replication accuracy The five E.coli
DNA polymerases Other enzymes are needed too The
stages of bacterial replication Membrane bound
replisomes in bacteria Eukaryotic replication
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The number of proteins involved in DNA metabolism
highlights the importance of this process
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Meselson-Stahl experiment Evidence for
semiconservative replication
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Visualization of bidirectional DNA replication
(? structures)
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5?3 and semidiscontinuous
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E.coli1 mistake in 109-1010 nucleotides 1
error in 1000-10,000 cell divisions Derived from?
Basepairing, proof reading, and repair
processes. Basepairing contributes to
replication fidelity
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Proofreading contributes to low observed levels
of mistakes. .but still does not get to in
vivo observations
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Variations in processivity correlate with
biological roles
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Nick translation DNA repair and removal of RNA
primers
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DNA Pol I, Kornberg, Nobel Prize and Cairns 1969
experiment.. Bacterial DNA polymerase III Many
enzyme activities are necessaryhelicase,
topoisomerase, DNA binding proteins, ligases
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ß subunits of E.coli DNA Pol III
1000 nt/s 100 rev/s 6000 rpm
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Initiation phase of DNA replication OriC 245 bp
containing short repeats three A/T rich 3mers
and four 9mers DnaA protein binding to 9mers
causes ATP-dependent unwinding of 13mers (A/T
rich) requires histone like HU protein DnaC
loads DnaBhelicases lead to bidirectional
unwinding Single stranded DNA binding protein
(SSB) plus TopoII lead to unwinding of
thousands of bp
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245 bp containing short repeats 3 A/T rich 3mers
and 4 9mers DnaA protein binding to 9mers causes
ATP-dependent unwinding of 13mers (A/T rich)
requires histone like HU protein DnaC loads
DnaBhelicases lead to bidirectional
unwinding Single stranded DNA binding protein
(SSB) plusTopoII lead to unwinding of thousands
of bp
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Dam (DNA adenine methyltransferse, GATC, 11
sites, cycling between hemi- and doubly
methylated, sequestering
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Elongation phase of replication and Okazaki
fragment synthesis
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Leading and lagging strand DNA synthesis
(opposite directions)
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Appearance of leading/lagging strand synthesis
going in the same direction through the single
DNA Pol III core
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Connecting the pieces DNA ligase
Viral and eukaryotes have ATP-dependent ligases
bacteria use NAD
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Replication termination Ter sequencesmultiple
copies of 20 bp element Tus protein
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Separation of catenanes done by a Topo II (Topo
IV)
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Chromosome partitioning (bacteria) Single
replication site (vs eukaryotes)
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Different types of polymerases Eukaryotes (a, d,
e etc.) Acyclovir Gertrude Elion Prodrug
(thymidine kinase, virally encoded) Selectively
inhibits herpes encoded DNA polymerase
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25.2 DNA repair Mutations and cancer Other
mechanisms are needed to explain the
data Redundancy in cellular repair
pathways Diverse repair pathways and enzyme
mechanisms
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Ames test (Bruce Ames (UCB)) Salmonella
typhimurium, his mutation auxotroph
Epigenetic basis of cancer E.g., Brca gene,
familial breast caner vs sporadic, mutational
forms
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One Renegade Cell (Paperback) by Robert A.
Weinberg "Cancer wreaks havoc in almost every
part of the human body... 
11.60
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Methylation and mismatch repair End counting in
eukaryotes?
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MutH recognizes GATC MutS recognizes mismatch
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Base excision pathway DNA glycosylase, AP
endonuclease, DNA Pol I, DNA ligase
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Nucleotide-excision repair (E.coli and humans)
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Repair of pyrimidine dimers with
photolyase direct repair Photolyase not found
in placental mammals
Xeroderma pigmentosum, or XP, is a genetic
disorder of DNA repair in which the body's normal
ability to remove damage caused by ultraviolet
(UV) light is deficient. This leads to multiple
basaliomas and other skin malignancies at a young
age. In severe cases, it is necessary to avoid
sunlight completely.
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Another example of direct repair
O6-methylguanine DNA methyltransferase
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What happens if not repaired?
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DNA recombination Homologous genetic
recombination Site-specific recombination DNA
transposition Transposable elements Function? DN
A repair, replication, regulation, chromosome
segregation, genetic diversity, embryonic
development
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Meiosis in eukyarotic germ-line cells Diploid
germ-line cells ? haploid gametes
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Homologous recombination Repair, physical link
during segregation, genetic diversity
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Recombination during meiosis
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How branch migration actually works.
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Enzymes that carry out recombinationcomplex E.col
i.recB, recB, recC and recA (genes)
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Chi 5GCTGGTGG3 1009 within E.coli genome
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RecA nucleoprotein filament on single stranded
DNA (EM)
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Six RecA subunits/turn, 24 subunits shown here
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Recombinational DNA repair of stalled replication
forks Unrepaired DNA lesions/nicks cause
problems
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RecF, RecO, RecR proteins or RecBCD complex
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Site specific recombination Recombinase Unique
20-200 bp sequences Integrase example
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Cre recombinase
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Effects of site-specific recombination
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Bacteriophage lambda integration/excision into
bacterial chromosome Phage encoded
recombinase XISbacteriophage, FIS bacterium
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Transposons, transposition, insertion sequences,
complex transpsosons.
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Direct and replicative transposition
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Recombination of V and J gene segments in human
IgG kappa light chain (antibody)
generation Millions of antibodies Only have
35,000 genes IgG class of immunoglobins Combinati
on of one V segment and one J segment during stem
cell differentiation 300 V element, 4 J 1200
possible V/J combos.occurs via
recombinations
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