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What happens to chromatin during replication

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What happens to chromatin during replication &/or gene expression (transcription) ... Other Chromatin-Associated proteins - DNA Polymerases - ssDNA binding proteins ... – PowerPoint PPT presentation

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Title: What happens to chromatin during replication


1
What happens to chromatin during replication /or
gene expression (transcription)?
  • Some stretches of the gene (promoter region) are
    devoid of histones (nucleosomes) during active
    gene expression
  • BUT
  • Nucleosomes are present on the rest of the gene.
  • -Promoter regions are hypersensitive to DNAse I
    (sequence independent endonuclease)

Gene
Promoter
2
  • Histones are NOT displaced by RNA Polymerase
  • During DNA Replication,
  • - Old histones are believed to remain with the
    leading strand
  • - New histones are thought to associate with the
    lagging strand

lagging strand
5
3
leading strand
3
Non-Histone DNA Binding Proteins Other
Chromatin-Associated proteins
- DNA Polymerases - ssDNA binding proteins -
Topoisomerase I II - Helicase - Primase, etc. -
RNA Polymerases - Transcription Factors -
activators suppressors - Factors for ribosomal
assembly - ribosomal proteins - Nuclear RNPs
(RiboNucleoProteins) involved in mRNA
processing - Telomere associated proteins -
Centromere associated proteins - Scaffolding
proteins - Recombination Proteins - Other
Replication
Transcription
RNA Processing
Others
4
Chromosome Structure, Function Transmission
Basic structure of eukaryotic chromosomes
Centromere
Telomere
Telomere
Telomere
Metaphase Chromosome
Centromere ?
Replication - Multiple Origins of replications -
Eukaryotic chromosomes are too large to replicate
from ONE origin of replication.
It would require over ten days to replicate a
chromosome with one ORI.
5
To be fully functional a eukaryotic chromosome
needs - at least one Origin of replication - a
centromere - telomeres 1. Origin of Replication
in YEAST ARS Autonomous Replication
Sequence 11 base ARS consensus
sequences 5-(A/T)TTTAT(A/G)TTT(A/T)-3 This
sequence is repeated multiple times in a 100 bp
region
6
2. Centromere - a unique sequence, usually (but
not always) at the midpoint of the chromosome
- The attachment site for spindle fibers in
MEIOSIS and MITOSIS. - Binding site for
kinetochore proteins
spindle fiber
7
  • 3. Telomere
  • - Highly repetitive sequences
  • DNA sequence that stabilizes the ends of
    chromosomes
  • - 3 OH is unprotected ? susceptible to
    exonucleases
  • Helps to solve the problem of replicating the
    ends of a linear DNA molecule.
  • - RNA primer required for DNA replication. How
    to put DNA at the new 5 end?

Confocal microscope image of chromasomes using a
general DNA probe (BLUE) along with telomere
specific probes (PINK).
8
- So after every round of DNA replication, the
chromosomes will get a little shorter -Unless
some other process occurs to lengthen the ends
9
  • How telomeres accomplish these tasks?
  • Telomeres are repetitive Guanine-rich sequences
    that often contain a 3 overhangs
  • - These sequences can fold back to form unusual
    GG base pairs Guanine Quartets
  • (G-quartet)
  • - These structures or the single stranded
    overhang itself may be capped by proteins to
    stabilize the ends

GG


GG
5
TTTTGGGGTTTTGGGG-3
10
(No Transcript)
11
II. An enzyme, telomerase, uses an RNA Template
(part of the enzyme) to synthesize new telomere
sequences on the ends of existing telomeres
12
Review of Lecture 9
  • DNA components
  • Sugar, phosphate, base
  • Nucleoside, nucleotide
  • Purines/pyrimidines
  • Polarity of strand
  • Glycosidic bond
  • Phosphodiester bond
  • DNA base pairing
  • AT versus CG
  • Hydrogen bonds
  • DNA structure
  • B form, A and Z
  • Properties of B DNA
  • Factors affecting denaturation

13
Review of Lecture 10
  • DNA forms
  • Circular vs. linear
  • Supercoiling
  • Closed circular DNA
  • Topoisomers
  • Twist, writhe and linking number- how are they
    related?
  • Topoisomerases
  • Differences between actions of topo I and Topo
    II
  • Replication
  • Meselson-Stahl experiment
  • Understand experiment and what principles it was
    based on

14
Review of Lecture 11
  • Replication
  • Required components
  • 5-3 direction
  • Replication fork
  • Leading, lagging strand
  • Lagging strand synthesis events
  • Primase
  • DNA pol III-extension
  • DNA pol I- nick translation
  • DNA ligase
  • Origin of replication
  • Single vs. multiple
  • Polymerases
  • Require a primer
  • Extension of new strand always in 5-3 direction
  • Pol I (Klenow fragment)
  • 5-3 exo activity
  • Degrades ds DNA
  • 3-5 exo
  • Degrades ss DNA
  • polymerase

15
Review of Lecture 12
  • DNA pol III
  • Holoenzyme core has polymerase and 3-5 exo
    activity
  • Accessory proteins- clamp loader and sliding
    clamp used for attaching pol to DNA
  • Eukaryotes have multiplepolymerases that carry
    out different fxns
  • Other proteins
  • SSBs
  • Bind to ss DNA
  • Helicases
  • Unwinds DNA strand
  • Binds to ss DNA
  • Topoisomerases
  • Topo I
  • Topo II/Gyrase

16
Review of Lecture 12- contd
  • Restructuring
  • DNA methylation
  • Bacteria
  • N6 methyladenine (mA)
  • Protection against viruses
  • Eukaryotes
  • 5-methylcytosine (mC)
  • may play a role in gene activation

17
Review of Lecture 13
  • Repair
  • De-amination of mC
  • Leads to CG?TA conversion since C is converted to
    T
  • Direct repair
  • Damaged bases not removed but repaired
  • Thymine dimers
  • photoreactivation
  • Alkylation of bases
  • G ?O6-alkyl guanine can bind to T instead of C
  • GC ?AT
  • Other types of repair
  • excision of thymine dimers by exonucleases
  • Base excision removes just the mutant base
  • Recombinational repair
  • Recombination allows undamaged strand to be used
    as template
  • SOS repair
  • Used in periods of high stress

18
Review of Lecture 14-15
  • Plasmids
  • Self replicating, circular DNA
  • One ori
  • Can carry drug resistance genes (useful for
    cloning purposes)
  • Bacterial viruses
  • Phage T4 most typical
  • Only DNA enters cells
  • Genome encodes proteins needed for packaging of
    new virus
  • Life cycle of lytic T4
  • Eukaryotic viruses
  • RNA genome
  • Ss or ds
  • DNA genome
  • Ss or ds
  • Linear or circular

Viruses Small, enclosed by a coat Vary in type
of DNA structure of coat mode of entry mechanism
of replication
19
Review of Lecture 14-15
  • Viruses and cancer
  • DNA viruses
  • Can cause cells to grow abnormally
  • RNA viruses
  • Infection leads to permanent genetic change due
    to integration
  • Retroviruses
  • Use reverse transcriptase (DNA pol) to make DNA
    copy, it integrates into host chromosome
  • After integration get transcription and synthesis
    of new genomes and components of viral particle
  • Viral replication
  • RNA viruses
  • - strand
  • Must contain replicase
  • strand
  • Coding strand, use host to synthesize replicase
  • DNA viruses
  • Requires a primer!
  • Begins at ori
  • Linear ss DNA genomes use terminal repeats

20
Review of Lecture 14-15
  • Viral budding
  • Assembly of viral particles through budding of
    host membrane with viral proteins
  • HIV
  • Retrovirus
  • Targeted for therapy with nucleoside analog and
    acyclovir
  • Transposable elements
  • mobile DNA that cannot leave host cell
  • Often encode transposase
  • Retrotransposons (same mechanism as retroviruses)
  • move through inverted repeat sequences
    recognized by transposase

21
Review of Lecture 16-18
  • DNA purification
  • Separate DNA away from proteins, etc.
  • electrophoresis
  • Restriction enzymes
  • Palindromic sequences cut ds DNA
  • Ligase forms phosphodiester bond
  • Kinases add phosphates to 5 ends
  • Plasmids
  • Used as vectors to carry foreign pieces of DNA
  • High copy number, easy to purify
  • Multiple cloning site
  • Cloning
  • Selecting transformants
  • Relica plating

22
Review of Lecture 16-18
  • cDNA
  • Represents only expressed genes
  • Making a libraryusing RT
  • mRNA
  • mRNA/DNA hyrbid
  • cDNA
  • Labelling techniques
  • End labelling with kinase
  • Nick translation
  • Labels throughout strand
  • Restriction mapping
  • Needed for making a map of uncharacterized DNA
  • Order fragments obtained by cutting with
    different restriction enzymes
  • Double digests critical!

23
Review of Lecture 16-18
  • Genomic library construction
  • Isolate genomic DNA
  • Partial cutting
  • Clone into appropriate vector
  • Comparison of cDNA vs. genomic library
  • PCR
  • Allows amplification of small amounts of DNA
  • Must know something about sequence
  • Steps in reaction
  • Site directed mutagenesis
  • M13 vector
  • Use oligonucleotide with mutation as primer for
    DNA pol

24
Review of Lecture 16-18
  • Sequencing
  • Maxam and Gilbert
  • Used chemicals to cut template strand, sequence
    on gel is template sequence
  • Sanger dideoxy
  • Chain terminators
  • Sequence run on gel represents newly synthesized
    strand that is complementary to template strand
  • Can be fully automated using fluorescent tags
  • DNase footprinting
  • Uses ds DNA cleavage to map protein binding sites
    on DNA
  • Blotting techniques
  • Based on nucleic acid hybridization or
    protein-antibody recognition
  • Plaque hybridization

25
Review of Lecture 16-18
  • Expression screening
  • Can make phage or bacteria conaining cloned DNA
    express the protein that DNA encodes
  • Can use hybridization to detect those specific
    proteins by probing with an antibody
  • Southern blotting/mapping
  • Combines restriction mapping with blotting
  • Requires making probes with certain pieces of DNA
    and determining what sequences they hybridize
    with

26
Review of Lecture 19-20
  • Nucleus
  • Has various compartments and specific structures
  • Nucleolus is site of ribosome assembly
  • Chromatin
  • DNA/protein complex
  • Levels of organization
  • Nucleosome
  • 30 nm fibre
  • loops
  • Nucleosome
  • Made up of 8 histones and 146 bp of DNA
  • Histone H1 clamps DNA onto nucleosome,
    participates in assembly of 30 nm fibre
  • Histones and replication

27
Review of Lecture 19-20
  • Centromere function
  • Telomere function
  • Replication of linear DNA ends
  • telomerase

28
Midterm exam is next Monday
  • There will be no class this Friday
  • Good luck!!
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