DNA Structure and Replication
• Pollard & Earnshaw Ch 12-14, 40-42
• Structure
– Chemical composition
– Filament packing
– Chromosome organization
• Replication
– Origin of replication complex
– Licensing
– MCM/polymerase
Project part 1
• Objective: construct an integrative cellular
model of tissue function, incorporating the
sensor and control systems presented in
class.
• Part 1: Identify an interesting tissue
– Anatomical description (anatomy, cell types)
– Functional description & control
– Quantitative assays for function
– Normal and pathological ranges for assays
• Sept 11
Sugar backbone
• Pentose sugar (deoxyribose)
• 5’ Phosphate
• Nitrogen rich base
C
O
C
OH
C
OH
C
OH
C
OH
Ribose
Base
C
OPO3
O
C
C
C
C
OH OH
deoxyribonucleotide
Adenosine
Pyrimidine
• Single nitrogen rich ring
– Planar bases due to N and O double bonding
Thymidine
Cytidine
Purine
• Double N-rich ring
– Also planar
– Base pair are asymmetric
Adenosine
Guanosine
Base Pairing
• Hydrogen bonds define pairing
T
A
G
C
Macromolecular conformation
• Sequence effects
• Accessory proteins
– Chromatin
– Chromosome
• Covalent modification
• Specialized domains
– Telomere
– Centromere
G-band ideogram
karyotype of human
chromosomes shows
distinct bands
Chromatin: Nucleosome
• Histone octamer
– Positively charged, attract negative phosphates
– Subject to extensive modification
• DNA
– 166 BP
– Twice wrapped
30 nm fiber
• Bead-on-string 10 nm fiber
– 166 BP nucleosome
– 34 BP linker DNA
– Histone H1
• Condenses to 30 nm
fiber
– 11nm helix
– 6 nucleosomes per turn
– H4 (deacetylated)
Dorigo et al., 2004
Heterochromatin
• Transcriptionally inert
• Highly condensed
• Gene inactivation
– Barr body
– CG methylation
Further condensation
• 100-300 nm fiber
• Domain loop model
– 50-100 kb
– Scaffold/Matrix Association
Region
• AT rich
• Base unpairing region
– Nuclear Matrix
• DNA topoisomerase: DNA knots
• Condensin: supercoiling
Centromere
• Chromatid pairing
• Kinetochore
• CEN sequence
– AT and AG Satellites
– Epigenetic modification
– Unidentified in mammals
• CENP proteins
Telomere
• Special structure to differentiate from strand
break
– 600-2500x 5’-CCCTAA-[…]-TTAGGG-3’
– 200 unpaired base overhang
• Prevent chromosomal erosion
– Telomerase elongates 3’ DNA
• Built-in RNA primer
• Active in intestines, testis, cancers
– Replicative senescence
Telomere structure
• Telomere Repeat Factors
(TRF)
• Ku capping protein (yeast)
– Strand repair function
• Loop formation
– Mammalian
• Physical distribution
Generic scheme of templatemediated synthesis
•
•
•
•
•
Identify the start site
Assemble the synthetic machinery
Wait for an initiation trigger
Synthesize
Stop/clean-up
Isolation of DNA polymerase
• Arthur Kornberg, 1959 Nobel Prize
– Work in 1956-1958 with Maurice Bessman,
Ernest Simms, I.R. Lehman and Julius Adler
– “Vital” processes vs chemical processes
– Eduard Buchner, 1907
• Cell-free synthesis of DNA
– DNA + cell extract  dinucleotides
– NTP + DNA + cell extract  dinuc +NTP
Cell free synthesis of DNA
• Highly optimized system
– E coli (doubling time 20 minutes)
– Massively radioactive NTP
– Very short incubation
• Assay conversion of acid-soluble NTP to
acid-insoluble DNA
• NTP + DNA + cell extract 0.0005% DNA
Cellular fractionation
• Start with 60L E coli culture 500 g cells
• Lyse and extract 40 g protein
– Synthesizes 1 nmole DNA/30 min/mg
• Mass/density separation
• Size separation
• Streptomycin precipitation
– Precipitates DNA with associated proteins
– Extract 2.5% protein
– Synthesizes 43 nmole DNA/30 min/mg
Cellular fractionation
• DNAse digest
– Solubilize DNA-bound protein
– 65% protein remains soluble (1.6% of total)
– Synthesizes 67 nmole
• Alumina gel precipitation
– Protein polarity, ala chromatography
– Collect 25% of DNAse fraction (0.4% total)
– Synthesizes 200 nmole/30 min/mg
• Two further fractionations
– Around 0.02% starting protein (8 mg)
– 2000 nmole/30 min/mg (~20% total activity)
Enzymatic properties
• Synthesizes DNA from diverse templates
– Bacterial, plant, mammal
– DNA is fundamentally identical
• Product has same dinucleotide composition
– DNA is a template, not a primer
• Faster on denatured DNA
– ie: single stranded
– Further validated Watson & Crick “template”
• Requires long strand template
• Works much better on phage than genomic
DNA Synthesis
• DNA polymerase
• Deoxyribose 3’ hydroxyl “attacks” nucleoside
triphosphate
Base
C OPO
3
O
– Forms phosphodiester bond
– Displaces HP2O73– Never backwards
• Okazaki fragments
C
C
C
C
OH
O
C OPOPO3PO3
O
Base
O
C
C
C
C
OH
DNA Polymerase
Template strand enters
polymerase
dsDNA exits
Pocket for NTP entry
http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?uid=69181
DNA Replication
•
•
•
•
•
•
•
•
Origin of Replication Complex (ORC) anneals to origin
ORC recruits Mini Chromosomal Maintenance (MCM)
MCM recruits Cdc45p
Cdc45p recruits DNA polymerase a/primase complex
Replication Factor C (RFC) displaces pola
RFC recruits Proliferating Cell Nuclear Antigen (PCNA)
PCNA recruits pold
DNA ligase stitches DNA fragments together
Origin of Replication
• Prokaryotes
– Single, circular DNA ~4,000,000 bp
– Replicator/Autonomously Replicating Sequence
• Identification by restriction fragment selection
Moore, et al.. Construction of chimeric phages and plasmids containing the origin of
replication of bacteriophage lambda. Science (1977) 198:1041-6.
– DNA synthesis 1000 bp/s or 65 min replication
• Simple Eukaryotes (S cerevisiae)
– 16 chromosomes, 107 bp genome
– Replicase 1000 bp/min or 10 hour !? replication
– ARS
• ~400
• 150 bp consensus sequence
Origin of Replication
• Multi-cellular Eukaryotes
– 2-D electrophoretic fractionation (fig 42-14)
– Initiation Zone
– Epigenetic mechanisms
• Proteins and mechanisms are highly
conserved
• Kornberg’s templates had to be long in order
to include an ORI
Pre-replication complex
• Origin Recognition Complex
– Assembles at origins during G1
– Orthologous to E Coli DnaA
• Cell Division Control 6+Cdt1
– Inhibitory complex
– E Coli DnaC
• Mini-Chromosomal
Maintenance proteins
– Recruited by Cdc6+Cdt1
– Licensing agent
– E Coli DnaB
Sun & al NSMB 2013
Transition to Replication
• Cdc45p anneals to ORC & Mcm
– Recruit GINS (Sld5, Psf1, Psf2, and Psf3)
– Activates Mcm helicase
– Recruits DNA polymerases and RPA
• RPA ssDNA binding protein
Leading strand
Lagging
strand
Simon & al Nature (2014)
Synthesis
• Polymerase a/Primase
• Polymerase d & e
• Proofreading
– DNA polymerase error rate ~1:104-105
– Human genome is 3 109 bases ~10,000 errors per
mitosis.
– Proliferating Cell Nuclear Antigen (PCNA) error
correction 1:109 bases, 3 errors per mitosis
– 1013 cells or 43 divisions => 130 base errors per adult
genome
• Okazaki fragment
• Topoisomerase
Summary
Cdc6 is believed to remain associated with MCMs
Fig 42-11