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
AdenosineC
C
C
C
C O
OH
OH
OH
OH
Ribose
deoxyribonucleotide
CC
C C
CO
Base OPO3
OH OH
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
A C
GT
• Hydrogen bonds define pairing
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 template-mediated 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– Forms phosphodiester bond
– Displaces HP2O73-
– Never backwards
• Okazaki fragments
CC
C C
CO
Base OPO3
OH
CC
C C
CO
Base OPO
OH
PO3PO3
O
O
DNA Polymerase
Template strand enterspolymerase
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 /primase complex• Replication Factor C (RFC) displaces pol• RFC recruits Proliferating Cell Nuclear Antigen (PCNA)• PCNA recruits pol• 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 selectionMoore, 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
Simon & al Nature (2014)
Leading strand
Laggingstrand
Synthesis
• Polymerase /Primase• Polymerase • 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
Fig 42-11
Cdc6 is believed to remain associated with MCMs