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What happens to chromatin during replication


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

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)

  • 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
leading strand
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
RNA Processing
Chromosome Structure, Function Transmission
Basic structure of eukaryotic chromosomes
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.
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
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
  • 3. Telomere
  • - Highly repetitive sequences
  • DNA sequence that stabilizes the ends of
  • - 3 OH is unprotected ? susceptible to
  • 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).
- So after every round of DNA replication, the
chromosomes will get a little shorter -Unless
some other process occurs to lengthen the ends
  • 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


(No Transcript)
II. An enzyme, telomerase, uses an RNA Template
(part of the enzyme) to synthesize new telomere
sequences on the ends of existing telomeres
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

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

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

Review of Lecture 12
  • DNA pol III
  • Holoenzyme core has polymerase and 3-5 exo
  • 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

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

Review of Lecture 13
  • Repair
  • De-amination of mC
  • Leads to CG?TA conversion since C is converted to
  • 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

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
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

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
  • 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

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

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!

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

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

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

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

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

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