NUCLEIC ACIDS REMEMBERED

TRANSFORMATIONDefinition: process in which genetic characteristics of an organism are

changed due to the absorption of DNA from a lysed bacterial cell.

Griffith’s experiment: proved transformation occurs but did not identify the “transforming agent”

AVERY, MACLEOD, MCCARTY

Series of experiments to determine transforming factor;

a) Mixed live non-virulent cells with different cell parts individually; only DNA caused transformation

b) Used combinations of DNA components- NO TRANSFORMATION

c) Ribonucleases & proteases – no effect on transformation; deoxyribonucleases inhibited transformation

Conclusion: DNA (whole molecule) transforming factor

Bacteriophagevirus that infects bacterial cells

Lederberg & Zinder

TRANSDUCTION Transmission of

genetic material by a virus

Resistant bacteria receive a new gene, transferred by the virus (from 1st host)

HERSHEY & CHASERadioactive isotopes (sulfur & phosphorus): prove that the DNA of

the phage enters the cell, protein coat remains outside the cell

LEDERBERG & TATUMConjugation – sexual reproduction in bacteria,

leads to genetic recombination

• 2 strains of DNA grown together – one has traits (A,B,C) other has (D,E,F)

• Cytoplasmic bridge (pilus) forms and cells exchange plasmid

• Offspring (recombinants) have traits of both parents (A,B,C,D,E,F)

DNA ERWIN CHARGAFF: analyzed nuclei of many

species Base pairing rules (1:1 ratios) Concentration of cytosine & guanine equal Concentration of adenine & thymine equal

ROSALIND FRANKLIN & MAURICE WILKINS‒ X-ray diffraction

WATSON & CRICK‒ DNA Model‒ Proposed semi conservative replication

• Double helix• Double strand of

nucleotides (deoxyribose, phosphate, nitrogen base) held together by H-bonds

• Anti-parallel strands• Purines: adenine &

guanine (double rings)• Pyrimidines: thymine &

cytosine (single rings)

DNA STRUCTURE

NOTE: # of H-bonds between bases, measurements, anti-parallel strands

DNA REPLICATION

DNA REPLICATION

DNA REPLICATION

DNA REPLICATION

DNA REPLICATION

Given one strand of DNA, what is the base sequence of the complimentary strand?

ACGTTGCAAGCTGACCTGGTCAG

REPLICATION MODELS

MESELSON & STAHL PROVE SEMICONSERVATIVE

REPLICATION

MESELSON & STAHL PROVE SEMICONSERVATIVE

REPLICATION

DNA has two “heavy” strands

DNA is now hybrid; ½ heavy, ½ light

MESELSON & STAHL PROVE SEMICONSERVATIVE REPLICATION

Conservative replication proven wrong.

Semi-conservative & dispersive still possible (all strands hybrids)

MESELSON & STAHL PROVE SEMICONSERVATIVE REPLICATION

After another replication (on “light” medium), semi-conservative replication confirmed (1/2 hybrid & ½ light)

Predict the next generation!

DNA replication:- DNA polymerases catalyze the reaction- Hydrolysis of phosphate bonds provides energy

DNA: anti-parallel strands

• Carbons of deoxyribose numbered 1' - 5'

• Phosphodiester bonds involve the 3' & 5' carbons

• One strand runs 5' to 3'• The other strand runs 3'

to 5'

1. DNA polymerase elongates DNA strands only in the 5' to 3'direction

2. One new strand, the leading strand, can elongate continuously 5' to 3'as the replication fork continues.

3. The other new strand, lagging strand, grows discontinuously in an overall 3' to 5' direction by adding short Okazaki fragments that are built in a 5' to 3' direction.

4. Ligase connects the Okazakifragments.

Priming DNA Synthesis

• Polymerase cannot initiate synthesis, it can only add to the end of an already started strand.

• Primase builds RNA nucleotides into a primer.

• RNA primer eventually replaced by DNA nucleotides

(topoisomerase)

Summary of DNA Replication

Ligasejoins Okazakifragments

Lagging strand-discontinuous synthesis –Okazaki fragments

Helicase unwindsparental double helix

Topoisomerase stabilizes unwound DNA

Leading strand, continuous synthesis

• DNA REPLICATION & MAINTENANCE• DNA Polymerase: enzyme which synthesizes single

DNA strand from template DNA (replication)• Whole nucleotides are bonded to complementary

nucleotides to form each new strand.– Trinucleotides are raw materials (ATP, GTP, TTP, CTP)– 2 (high energy bonds) used to accomplish bonding

(energy expensive); AMP, GMP,TMP,CMP bonded to each other by DNA polymerase.

• Other enzymes involved in maintaining DNA structure.– Recognition enzymes (proof reading enzymes) scan DNA

molecule to identify atypical or injured DNA– Endonucleases (restriction enzymes) – breaks DNA above

& below “atypical” sites.– DNA polymerase – synthesizes single strand segments to

replace “damaged” segments.– DNA ligase – binds new segment to old strand.

ENZYMES WHICH MAINTAIN DNA

•“Scanner” or proofreading enzyme checks DNA for damage

•Endonuclease (restriction enzyme)cuts DNA

•DNA Polymerase adds new nucleotides

•DNA Ligasejoins new nucleotides (S-P)links Okazaki fragments

The end-replication problem:

Gap left at the 5’ end of each chromosome.Each end gets shorter with every replication

Telomeres-short nucleotide sequences at the end of each chromosome.- protect the genes- telomerase, present in germ cells, produces telomeres

Humans: TTAGGG