chapter 16 molecular basis of inheritance (dna structure and replication) helicase enzyme

Chapter 16Chapter 16

Molecular Basis of Inheritance

(DNA structure and Replication)

Helicase Enzyme

What is the genetic material? DNA or protein?

The Amazing Race

1928 Griffith – transformation of pneumonia bacterium

1944 Avery – further studied

transformation by destroying lipids,

CHO, and proteins

1947 Chargaff –

• Quantified purines and pyrimidines

• Suggested base pairing rules (A=T, C=G)

1950 Wilkins and Franklin – DNA X-rays

(a) Rosalind Franklin (b) Franklin’s X-ray diffraction

photograph of DNA

1952 Hershey and Chase – bacteriophages – incorporation of

radioactive viral DNA in new phages

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

Empty protein shell

Phage DNA

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

Empty protein shell

Phage DNA

Centrifuge

Centrifuge

Pellet

Pellet (bacterial cells and contents)

Radioactivity (phage protein) in liquid

Radioactivity (phage DNA) in pellet

1953 Watson and Crick – DNA Model

1962 Nobel Prize awarded to Watson and Crick and Wilkins

** Conclusion: DNA = Genetic Material, not Protein

Models of DNA Replication

Semi-Conservative Model(1950s - Meselson and Stahl)

Fun DNA Replication Facts

• 6 billion bases in human cell = 2 hours of replication time

• 500 nucleotides added per second

• Accurate (errors only 1 in 10,000 base pairs)

Anti-Parallel

Structure of DNA

Mechanism of Replication Step 1

• Origins of Replication = Special site(s) on DNA w/Specific sequence of nucleotides where replication begins– Prokaryotic Cells =

1 site (circular DNA)– Eukaryotic Cells =

several sites (strands)

Steps 2 - 5• Helicase: (enzyme) unwinds

DNA helix forming a “Y” shaped replication fork on DNA

• Replication occurs in two directions, forming a replication bubble

• To keep strands separate, DNA binding proteins attach to each strand of DNA

• Topoisomerases: enzymes that work w/helicase to prevent “knots” during unwinding.

Step 6 - Priming

• Priming = due to physical limitation of DNA Polymerase, which can only add DNA nucleotides to an existing chain

• RNA primase – initiates DNA replication at Origin of Replication by adding short segments of RNA nucleotides.

• Later these RNA segments are replaced by DNA nucleotides by DNA Pol.

Step 7• DNA Pol. = enzyme that

elongates new DNA strand by adding proper nucleotides that base-pair with parental DNA template

• DNA Pol. can only add nucleotides to the 3’ end of new DNA, so replication occurs from a 5’ to 3’ direction

• Leading vs. Lagging Strand results

Leading vs. Lagging Strand

• Leading Strand: strand that can elongate continuously as the replication for progresses

• Lagging Strand: strand that cannot elongate continuously and moves away from replication fork.

• Short Okazaki fragments are added from a 5’ to 3’ direction, as replication fork progresses.

3’5’

3’ 5’

5’

3’

3’5’

Step 8• DNA Ligase = enzyme that “ligates” or covalently

bonds the Sugar-Phosphate backbone of the short Okazaki fragments together

• Primers are required prior to EACH Okazaki fragment

DNA i

Flash Overview

Step 10: Fixing Errors

• DNA Pol. Proofreads as it elongates

• Special enzymes fix a mismatch nucleotide pairs

• Excision Repair:– Nuclease: Enzyme

that cuts damaged segment

– DNA Pol. Fills in gap with new nucleotide

Mutations

• Thymine Dimers (covalent bonding btwn Thymine bases) –often caused by over-exposure to UV rays DNA buckeling skin cancer results, unless corrected by excision repair

• Substitutions: incorrect pairing of nucleotides• Insertions and Deletions: an extra or missing

nucleotide causes “frameshift” mutations (when nucleotides are displaced one position)

Problems with Replication

• Since DNA Polymerase can only add to a 3’ end of a growing chain, the gap from the initial 5’ end can not be filled

• Therefore DNA gets shorter and shorter after each round of replication

Solution?• Bacteria have circular DNA

(not a problem)• Ends of some eukaryotic

chromosomes have telomeres at the ends (repeating nucleotide sequence that do not code for any genes)

• Telomeres can get shorter w/o compromising genes

• Telomerase = enzyme that elongates telomeres since telomeres will shorten

Telomerases are not in most organisms

• Most multicellular organisms do not have telomerases that elongate telomeres (humans don’t have them)

• So, telomeres = limiting factor in life span of certain tissues

• Older individuals typically have shorter telomeres