Mb 414 Bacterial genetics 1

Mutation and repair lecutre

“classical” genetics

• best way to tell what a gene does: break(mutate) it and see what happens

  • look for phenotype

• best way to tell what gene(s) control a process: mutate lots of genes and look for an organism that can’t do the process

  • make mutant library- are there any strains in my library that can’t do that

Definitions:

• species - a population of mciroorgnisms with similar characteristics

• clone - a population of cells that are genetically identifical (strain)

• genotype - the specific set of genes present in a cell

• phenotype - the collecttion of characteristics that are observable

• wild type strain: a recogized “type”strain, which is a clonal population of a particular species, has the identical genotype

  • characterized - known by name

Point mutations

• original sequence→

  • silent - even though we have differnt codon - code for same amino acids

  • Missence - put stop codon - wipe protein

Insertions and deletions

• worse than point mutations

• adding/subtracting a nucelotide

• unless a multiple of three - may just add another or take away amino acid

• will make different protein

Spontaneous mutations

• very rare: less than 1×10³ per generation in e. coli

  • (0.001 SNPs per generation)

• causes

  • main cause: misincorporaton during DNA replication

  • DNA damage:

    • UV

    • ionizing radiation

    • depurination: base-sugar separation

    • oxidative damage

UV induced damage

• UV ligth (less thna 280nm) is mutagenic

• Causes dimerization of thymine

• strong UV light breaks DNA backbone

1. when light comes in and there are two adjacent pyrimidines (occurs on one strand)

2. Cyclobutane ring forms between two pyrimidines

3. Forms a cross linking between the two

4. pyrimidine thymine dimer will no longer going to base pair across

5. DNA replication does not recognize dimer as base

   1. will need fixing before replication

Ionizing radiation

• Radiation forms chemical free radical

• cause double stranded breaks in DNA

• can cause sugar-base breaks called depurination

1. Hits a base/sugar bond, → breaks

2. basewill float away (double helix with a hole)

3. DNA replication comes thorugh and base is missing

   1. fixed by liagse before replication

1. Hits phosphodiester backbone

2. double stranded break

3. results in death, no mechanims for fixing

Depurination

• invovles separation of base from suagr

• problems arrise during DNA replication

• can occur when base is in acidic conditions

Deamination

• Takes nitrogen group off and registers base as different nucleotide

• Recognizes C as a T

Oxidative damage

• Guanine in hydrogen peroxide is more susceptible to becoming 8-oxo-guanine

• oxidatively damaged guanine can occur in DNA and as a nucleotide

• causes shift in base pairing hydrogens (syn conformation → 2 H bonds vs 3H bond)

  • DNA polymerase places an A across from 8-oxoG

  • fixed as a mutation

DNA repair

• Responsible for the high fidelity of DNA replication and the low spontaneous mutation frequency

• fix errors due to misincorporation

• fix induced errors

Photolyase - pyrimidine dimers

• three methods to resolve dimers

  • light dependent photolyase enzyme

  • excision repair

  • recombination repair

• Photolyase is activated by visible ligth (340-400nm) to monomerize the dimer (photoreactivation)

  • removes crosslinks

Photolyase

Binds to DNA kink and activated by visible light (photoreactivation)

most common way to result dimers

Nucleotide excision repair

Mediated by uvrABCD complex

1. UvrAB detects error

2. UvrC is recruited to the error

3. UvrC then cuts the backbone 8 base 5’ to error and 4 bases 3’

4. UvrD (helicase) releases the 12 base region containing the eroor

5. The gap is filled and ligated

Methyl directed mismatch repair

• Takes advantage of that dam methylase, to repair misincorporated bases in fairly new DNA

• DNA replication is semi-conservative, the new strands will be hemi-methylated

  • keep origin from firing again

  • slow

  • helpful for errors

Methyl directed mismatch repair steps

• takes advantage of slow pace of dam methylase to repair mismatched bases

• methylated is older

• 1. MutS comes in and recognizes a base is wrong and binds

• 2. MutL and MutH are recruited to mismatch

  • helps determine which base is the right one

• 3. DNA gets looped thorugh until hemi-methylated dam site is encountered

• 4. mutH nicks the unmethylated strand (new strand)

• 5. DNA is unwound by UvrD and the ssDNA is chewed up

• 6. Gap is repaired by DNA polymerase and ligase

DNA glycosylases (Base excision repair)

• Cleave the sugar-base bind in altered or damaged nucleotides

• Uracil DNA glycosylase: removed demaminated cytosine

• Hypoxanthine DNA glycosylase: deaminated adenine

• FAPY glycosylase: removed8- oxoguanine

• The nick is filled by DNA polymerase and ligase

Daughter strand repair

• occurs at replication form when damage has yet to be fixed

  • DNA polymerase pauses at damaged base

• Goes through recombination process and adds copy of strand from the other new created daughter strand

Guest lecture