BIO 206 Lab Module 2

differences between S. cerevisiae and E.coli (including on microscope)

S. cerevisiae: yeast, single-celled eukaryote

• eukaryote=> bigger, have organelles

E.coli: bacteria, prokaryote

• prokaryote=> smaller

3 advantages of using yeast as model organism

1. single-celled=> easy to work with

2. homologous genes to humans

3. can exist in haploid or diploid state→ lets them mimic more advanced eukaryotes while keeping genetically recessive phenotypes easy to see

What is proofreading during DNA replication, and which enzymes can do this function?

Proofreading: identifies and corrects mistakes in DNA

Enzymes: DNA pols (ε and ɗ)

• ε: leading strand

• ɗ: lagging strand
  

3 types of DNA lesions and what they do

1. mutagenic: changes the sequence of the new DNA strand

2. cytotoxic: causes cell death

3. cytostatic: inhibits cell growth

endogenous damagedefinition

spontaneous damage

environmental damage definition

induced damage

2 major ways to deal with cell damage

1. DNA repair

2. DNA damage tolerance

Draw out the DNA damage response pathways

Important proteins in the Mismatch Repair (MMR) pathway

MLH1, MSH2, and PMS1

3 factors DNA replication is dependent on

1. Base selectivity of replicative DNA pols (how accurate they are)

2. proofreading

3. MMR

Purpose of DNA Pol Zeta in DNA replication and effects on mutations

purpose: adds base across from lesion and extend a few bases past the legion→ allows for Pol Delta/Epsilon to finish replication

effects: since Pol Delta/Epsilon can finish

3 measurements of yeast experiment

measuring:

1. Spontaneous Mutagenesis— how many CAN1 mutants rise from replication errors or endogenous DNA damage

2. Survival of cells after DNA damage from UV irradiation

3. UV induced mutagenesis— how many CAN1 mutants arise fro mutations caused by UV irradiation

Survival Equation

Survival= (# colonies on UV-treated SC plate)/(# colonies on SC plate with no UV)

Total Mutant Frequency Equation

Mutation Frequency= (# colonies on UV treated SC+ CAN plate)/(#colonies on SC plate wit same UV treatment x dilution factor)

Induced Mutation Frequency Equation

Induced Mutation Frequency= (mutation frequency at UV dose)— (Mutation frequency for plate with no UV)

Yeast Strains used with Level 1 Mutations

WT- Wildtype, no mutations (control)

PMS1△- Deletion of PMS1 gene

PR- Pol Epsilon can’t proofread

DD- Pol Zeta is nonfunctional

What yeast strain most likely to result in more level 2 mutants than the control?

PMS1△- PMS1 no longer helps in MMR

PR- Pol Epsilon can no longer check and fix mistakes (proofread) x