Chapter 16 The Molecular Basis of Inheritance

Question? Traits are inherited on chromosomes, but what in the chromosomes is the genetic material? Two possibilities: Protein DNA

Qualifications Protein: very complex. high specificity of function. DNA: simple. not much known about it (early 1900’s).

For testing: Name(s) of experimenters Outline of the experiment Result of the experiment and the importance of the result

Griffith Pneumonia in mice. Two strains: S - pathogenic R - harmless

Griffith’s Experiment

Result Something turned the R cells into S cells. Transformation - the assimilation of external genetic material by a cell.

Problem Griffith used heat. Heat denatures proteins. So could proteins be the genetic material? DNA - heat stable. Griffith’s results contrary to accepted views.

Avery, McCarty and MacLeod Repeated Griffith’s experiments, but added specific fractions of S cells. Result - only DNA transformed R cells into S cells. Result - not believed.

Hershey & Chase Genetic information of a virus or phage. Phage - virus that attacks bacteria and reprograms host to produce more viruses.

Bacteria with Phages

Phage Components Two main chemicals: Protein DNA Which material is transferred to the host?

Used Tracers Protein - CHONS, can trace with 35 S. DNA - CHONP, can trace with 32 P.

Experiment Used phages labeled with one tracer or the other and looked to see which tracer entered the bacteria cells.

Result DNA enters the host cell, but the protein did not. Therefore: DNA is the genetic material.

Picture Proof

Chargaff Studied the chemical composition of DNA. Found that the nucleotides were in certain ratios.

Chargaff’s Rule A = T G = C Example: in humans, A = 30.9% T = 29.4% G = 19.9% C = 19.8%

Why? Not known until Watson and Crick worked out the structure of DNA.

Watson and Crick Used X-ray crystallography data (from Rosalind Franklin) Used model building. Result - Double Helix Model of DNA structure. (One page paper, 1953).

Rosalind Franklin

Book & Movies “The Double Helix” by James Watson- His account of the discovery of the shape of DNA Movie – The Double Helix

DNA Composition Deoxyribose Sugar (5-C) Phosphate Nitrogen Bases: Purines Pyrimidines

DNA Backbone Polymer of sugar-phosphate. 2 backbones present.

Nitrogen Bases Bridge the backbones together. Purine + Pyrimidine = 3 rings. Constant distance between the 2 backbones. Held together by H-bonds.

Chargaff’s Rule Explained by double helix model. A = T, 3 ring distance. G = C, 3 ring distance.

Watson and Crick Published a second paper (1954) that speculated on the way DNA replicates. Proof of replication given by others.

Replication The process of making more DNA from DNA. Problem: when cells replicate, the genome must be copied exactly. How is this done?

Models for DNA Replication Conservative - one old strand, one new strand. Semiconservative - each strand is 1/2 old, 1/2 new. Dispersive - strands are mixtures of old and new.

Replication Models

Meselson – Stahl, late 1950’s Grew bacteria on two isotopes of N. Started on 15 N, switched to 14 N. Looked at weight of DNA after one, then 2 rounds of replication.

Results Confirmed the Semiconservative Model of DNA replication.

Replication - Preview DNA splits by breaking the H- bonds between the backbones. Then DNA builds the missing backbone using the bases on the old backbone as a template.

Origins of Replication Specific sites on the DNA molecule that starts replication. Recognized by a specific DNA base sequence.

Prokaryotic Circular DNA. 1 origin site. Replication runs in both directions from the origin site.

Eukaryotic Cells Many origin sites. Replication bubbles fuse to form new DNA strands.

DNA Elongation By DNA Polymerases such as DNA pol III Adds DNA triphosphate monomers to the growing replication strand. Matches A to T and G to C.

Energy for Replication From the triphosphate monomers. Loses two phosphates as each monomer is added.

Problem of Antiparallel DNA The two DNA strands run antiparallel to each other. DNA can only elongate in the 5’--> 3’ direction.

Leading Strand Continuous replication toward the replication fork in the 5’-->3’ direction.

Leading Strand 1. DNA helicase unwinds the DNA at the replication forks. -leading strand (3’-5’), replicates (5’-3’) towards the fork. 2. Molecules of single strand binding protein prevent the DNA from sticking back together. 3. Primase synthesizes an RNA primer at the end of 5’end. 4. DNA pol III synthesizes the strand continuously.

Lagging Strand 1. DNA helicase unwinds the DNA at the replication fork. -lagging strand (5’-3’) cannot replicate in the 3’-5’ direction, replicates away and towards the fork. 2. Primase joins RNA nucleotides into a primer. 3. DNA pol III adds DNA nucleotides to the primer forming an Okasaki fragment After reaching the next RNA primer DNA pol III detaches.

5. Fragment 2 is primed, then DNA pol III adds DNA nucleotides, detaching when it reaches the fragment 1 primer. 6. DNA pol I replaces the RNA with DNA, adding nucleotides to the 3’ end of fragment DNA ligase forms a bond between the newest DNA and the DNA of fragment This continues until the strand is replicated.

Priming DNA pol III cannot initiate DNA synthesis. Nucleotides can be added only to an existing chain called a Primer.

Primer Make of RNA. 10 nucleotides long. Added to DNA by an enzyme called Primase. DNA is then added to the RNA primer.

Priming A primer is needed for each DNA elongation site.

Lagging Strand Discontinuous synthesis away from the replication fork. Replicated in short segments as more template becomes opened up.

Okazaki Fragments Short segments ( bases) that are made on the lagging strand. All Okazaki fragments must be primed. RNA primer is removed after DNA is added.

Enzymes DNA pol I - replaces RNA primers with DNA nucleotides. DNA Ligase - joins all DNA fragments together.

Other Proteins in Replication Topoisomerase – relieves strain ahead of replication forks. Helicase - unwinds the DNA double helix. Single-Strand Binding Proteins - help hold the DNA strands apart.

DNA Replication Error Rate 1 in 1 billion base pairs. About 3 mistakes in our DNA each time it’s replicated.

Reasons for Accuracy DNA pol III self-checks and corrects mismatches. DNA Repair Enzymes - a family of enzymes that checks and corrects DNA.

Homework Read Chapter 16 (Hillis – 9) Be sure to check out the “Links” are under course content. Exam 1 – Tuesday 2/12 No broadcast Thurs. 2/14

DNA Repair Over 130 different DNA repair enzymes known. Failure to repair may lead to Cancer or other health problems.

Example: Xeroderma Pigmentosum - Genetic condition where a DNA repair enzyme doesn’t work. UV light causes damage, which can lead to cancer.

Xeroderma Pigmentosum Cancer Protected from UV

Thymine Dimers T-T binding from side to side causing a bubble in DNA backbone. Often caused by UV light.

Excision Repair Cuts out the damaged DNA. DNA Polymerase fills in the excised area with new bases. DNA Ligase seals the backbone.

Problem - ends of DNA DNA Polymerase can only add nucleotides in the 5’--->3’ direction. It can’t complete the ends of the DNA strand.

Result DNA gets shorter and shorter with each round of replication.

Telomeres Repeating units of TTAGGG ( X) at the end of the DNA strand (chromosome) Protects DNA from unwinding and sticking together. Telomeres shorten with each DNA replication.

Telomeres

Serve as a “clock” to count how many times DNA has replicated. When the telomeres are too short, the cell dies by apoptosis.

Implication Telomeres are involved with the aging process. Limits how many times a cell line can divide.

Telomerase Enzyme that uses RNA to rebuild telomeres. Can make cells “immortal”. Found in cancer cells. Found in germ cells. Limited activity in active cells such as skin cells

Comment Control of Telomerase may stop cancer, or extend the life span.

NEWS FLASH The DNA of Telomers is actually used to build proteins. These proteins seem to impede telomerase. Feedback Loop??