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

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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

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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

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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

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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

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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

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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
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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

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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

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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

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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!

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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

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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

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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

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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

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Review of Lecture 19-20

• Centromere function
• Telomere function
• Replication of linear DNA ends
• telomerase

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Midterm exam is next Monday

• There will be no class this Friday
• Good luck!!