BIOL2020 - Chapter 25 - DNA Replication and Repair

What were the 3 proposed methods of DNA replication and which one was accepted as true?

• Conservative

• Semi-conservative

• Dispersive

DNA replication is semi-conservative.

How was the correct method of DNA replication discovered?

By synthesizing DNA from a template using heavy isotopes and weighing the strands between rounds of replication

What reaction does DNA catalyze (write out reaction)?

dNTP + (dNMP)_n → (dNMP)_n+1 + PPi

What phosphates are removed when a nucleotide is added to a sequence by DNA polymerase?

β and γ

What is the major type of DNA polymerase in E. Coli

DNA Polymerase III

Why can incorrect base pairs not bond properly?

Because they do not have the right geomerty.

What part of a Polymerase fixes incorrectly place base pairs?

The exonuclease site.

How do Polymerases recognize mistakes in base pairing?

The shape, due to incorrect bonding.

What direction is the exonuclease site of a polymerase III that corrects mismatched base pairs in?

3’ to 5’

How does DNA Polymerase I use its 5’ to 3’ exonuclease site?

Removes DNA ahead of it by “nick translation” and add in DNA in its place behind it.

What enzyme creates the start of a strand of DNA during synthesis?

Primase.

Why is a primer necessary for DNA synthesis?

DNA cannot be built off of nothing, RNA can.

What enzyme replaces RNA primers with DNA?

DNA Polymerase I

What enzyme seals holes/nicks in DNA?

Ligase

What directions can Polymerase I and III move when on a strand?

Backwards and forwards.

Which Polymerase can remove sequences infront of it?

Polymerase I.

What is the difference between a transition and a transversion?

Both are single base pair substitutions; Transition goes from a purine to a purine or a pyrimidine to a pyrimidine, Transversion goes from a purine to a pyrimidine or vie versa.

How are mismatched base pairs fixed in E. Coli?

• MutL-Muts complex recognizes bulge caused by mismatch and binds

• MutH reels in DNA from both ends and cleaves the strand, forming a nick

• Nicked strand is degraded by exonuclease and filled in with DNA Polymerase III

• Process uses 2 ATP

When a mistake in base paring is detected in E. Coli, how can enzymes tell which strand has the mismatched base?

After DNA replication, new strand is unmethylated while old strand is (called hemimethylated)

MutH recognized the unmethylated strand as the new strand and cleaves it.

What is Base excision repair used for in E. Coli?

Removing accidentally placed/converted Uracil from DNA.

How is base excision repair in E. coli done?

• DNA Glycosylate breaks glycosidic bond between suagr and base and removes the base

• AP endonuclease breaks phosphodiester bond of nucleotide with no base and creating a gap

• DNA Polymerase I starts at the gap and removes and replaces nucleotides in front of it

• DNA ligase closes DNA strand

How does a cell repair a double stranded DNA break?

The cell uses the unbroken chromosome as a template to fix the break.

What can result from a double stranded DNA break if it is not fixed?

Apoptosis.