Chapter 15 - DNA synthesis and Repair

Hershey-Chase Experiment

Basis:

• DNA contains P but not S

• Protein contains S but not P

Evidence:

• Radioactive DNA is in pellet

• Radioactive protein is in solution

What is replication carried out by

DNA polymerase (enzyme)

• Makes a polymer of DNA

• Catalyzes DNA synthesis

DNA polymerase limitations

• DNA polymerase proceed ONLY 5’ → 3’

  • Must have free 3’-OH to attach incoming nucleotide

  • Must have second strand to use a template

DNA polymerase in bacteria

• Labeled with Roman numbers

  • DNA Pol. I-V

• Carry out replication, repair, or both

Where does the energy for synthesis come from?

dNTPS- Deoxyribonucleoside triphosphate

• each nucleotide being added brings its own energy

Origin of replication for bacteria? Eukaryotes?

Bacteria- one origin, one replication bubble

Eukaryotes- many simultaneous origins

Leading strand

“Continuous strand”

Lagging strand

“discontinuous strand”

Helicase

Opens double helix

Single Strand DNA-binding proteins

Binds to single-stranded DNA and keeps it from closing back up

Topisomerase

Straightens out double strands so helicase can easily separate them

Primase

RNA polymerase

• Does not require free 3’-OH to begin synthesis

• Provides the free 3’-OH for DNA Pol

DNA polymerase III

After RNA primer is established

• synthesizes leading strand in 5’→3’ direction

Okazaki fragments

• Short, newly synthesized DNA fragments formed on the lagging template

• Large piece of DNA, small piece of RNA

DNA polymerase 1

Removes RNA and builds new DNA in that spot

DNA Ligase

Glues the okazaki fragments together to make one complete strand of DNA

The end replication problem

Lagging strand

• when RNA primer is removed, DNA primase would typically add dna in its place

• however, they must have 3’OH to build from so there is a little piece that doesn’t get copied

Chromosomes will shorten after very round of DNA replication

Telomere

End of a linear chromosome

• Made of short, repeated sequences (TTAGGG)

Telomerase

Enzyme that extends telomeres

• solves the end replication problem by extending unreplicated strand

  • Gives more space for replication to happen

Limitations of telomerase

In limited cell types

• I humans, only gamete-producing cells

Somatic cells:

• They will stop dividing after a certain amount of cell divisions based on how long the telomeres are

What happens if cells produce overactive telomerase?

More DNA replication than you want to and more cell division than wanter

• Cancer possibly

DNA Repair (how often DNA polymerase is making a mistake)

DNA pol 3 error rate: ~1 in 100,000 bases

• correct base pairs are most energetically favorable

• correct base pairs have a shape distinct from incorrect

Actual #error found ~ 1 in 10^9 bases

Proofreading (repair mechanisms)

DNA pol III can detect and correct mismatched base pairs

• autocorrect

• Polymerase pauses when it detects a mismatch

• A separate subunit of polymerase has exonuclease ability

Mismatch repair

Corrected after DNA replication by specialized proteins

• letting a friend read your paper

Nucleotide excision repair

Removal and replacement of mismatched bases