F CH7 key concepts F genetic material: DNA F DNA structure F DNA replication LECTURE 12: DNA: STRUCTURE & REPLICATION I.

CH7 key concepts

genetic material: DNA

DNA structure

DNA replication

LECTURE 12: DNA: STRUCTURE & REPLICATION I

CHAPTER 7: KEY CONCEPTS transforming principle in bacteria is DNA experiments with labeled T2 phage establish that

DNA is the hereditary material Watson & Crick DNA model: anti-parallel double

helix = nucleotides linked by phosphodiester bonds; helices are held together by hydrogen bonds between pairs of nitrogenous bases

DNA structure ensures fidelity of replication; base pairing specified by hydrogen bonding

DNA replication is semiconservative; daughter duplex has 1 parental & 1 new strand

DNA replication depends on many enzymes

DNA = GENETIC MATERIAL Griffith (1928), Streptococcus pneumoniae transforming principle in bacteria is DNA

DNA = GENETIC MATERIAL Avery et al. (1944), Streptococcus pneumoniae more proof ... transforming principle is DNA

DNA = GENETIC MATERIAL Hershey & Chase (1952), T2 bacteriophage DNA (not protein) is genetic material

why was the scientific community reluctant to accept DNA (rather than protein) as hereditary material?...

DNA is a simple molecule...

how is complexity of life encoded by such simplicity?

how is information accurately transmitted from one generation to the next?

DNA = GENETIC MATERIAL

DNA STRUCTURE chemical structure of the 4 nucleotides in DNA

DNA STRUCTURE

hydrogen bonded nucleotides on opposite helices

DNA helices are antiparallel

carbons on sugar define ends... 5' and 3'

pyrimidines bond with purines T A C G

DNA STRUCTURE bonding is specific: pyrimidines bond with purines implication: mechanism for accurate replication

DNA STRUCTURE helical structure of DNA

major & minor groves

10Å radius & 20Å diameter

3.4Å between nucleotide base pairs

34Å / 360° turn

10 nucleotide base pairs / 360° turn

DNA STRUCTURE

clues for Watson & Crick (1953) ... X-ray data

& ratios ( A + G ) = ( T + C ) ... Chargaff

DNA STRUCTURE DNA double helix (space filling model)

several forms... A, B & Z DNA

DNA REPLICATION model of replication proposed

by Watson & Crick (1953)

parental strand = template

semiconservative model (new double helix has 1 template + 1 new daughter strand)

3 possible models: conservative semiconservative dispersive

DNA REPLICATION 3 possible models of DNA replication:

DNA REPLICATION Meselson & Stahl (1958), tested the models

DNA REPLICATION what would have happened if...

DNA REPLICATION plant root cells incubated with [3H] thymidine

colchicine to inhibit spindles (chromosomes do not separate)

autoradiography

DNA REPLICATION explanation of [3H] thymidine incubation / colchicine

treatment / autoradiography at DNA level

DNA REPLICATION “harlequin chromosomes”, incubated with

bromodeoxyuridine (BUdR)

DNA REPLICATION harlequin chromosomes of Chinese hamster ovary

cells, incubated with bromodeoxyuridine (BUdR)

chromatidexchange

DNA REPLICATION

semiconservative replication

linear gene arrangement

... chromatid = single DNA molecule + protein matrix

chromatidexchange

DNA REPLICATION model of replication proposed

by Watson & Crick (1953)

parental strand = template

semiconservative model (new double helix has 1 template + 1 new daughter strand)

DNA REPLICATION model of replication proposed

by Watson & Crick (1953)

parental strand = template

semiconservative model (new double helix has 1 template + 1 new daughter strand)

replication fork

DNA REPLICATION

replication fork ? bacteria incubated with [3H] thymidine... daughter molecules 1 “hot” + 1 “cold” strand autoradiograph...

DNA REPLICATION

replication... theta () structures

replication fork

DNA REPLICATION

some molecules use rolling-circle replication

MECHANISM OF DNA REPLICATION

Kornberg (late ‘50s)

enzyme that catalyzes DNA replication ... DNA polymerase I

several others

MECHANISM OF DNA REPLICATION

DNA replication requirements... 1. H bonds between bases must be broken 2. chain separation/unwinding 3. available pools of 4 dNTPs: A = T, C G 4. enzymes

E. coli... ~4.7 × 107 bp (47,000,000) molecule has ~ 4.7 × 106 turns (10 bp/turn) 1500 bp/s, ~ 20 min to divide with 1 origin

MECHANISM OF DNA REPLICATION prokaryotes have a single origin of replication bidirectional replication with 2 forks single terminus ~ 20 minutes (*... book says 40)

MECHANISM OF DNA REPLICATION E. coli origin of replication tandem repeat consensus sequence protein binding sites

MECHANISM OF DNA REPLICATION

replication to completion...

MECHANISM OF DNA REPLICATION eukaryotes have multiple origins of replication bidirectional replication with 2 forks 1.4 h (yeast) to 24 h (cultured animal cells)... or more issues...

linear vs circular coordination of multiple chromosomes

pulse/chase and autoradiography ...

MECHANISM OF DNA REPLICATION eukaryotes have multiple origins of replication bidirectional replication with 2 forks 1.4 h (yeast) to 24 h (cultured animal cells)... or more issues...

linear vs circular coordination of multiple chromosomes

pulse/chase and autoradiography ... Drosophila