DNA Structure and Replication

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