Chapter 6: DNA replication and repair

what does DNA repair do?

It repairs any damage or removes modifications on a DNA strand and returns it to its original state

What was the Meselson Stahl experiment?

An experiment that used centrifugation to determine the replication model for DNA

How was the Meselson Stahl experiment performed?

A 14N bacteria was grown in a light medium and produced a DNA band near the top of the centrifuge tube, while A 15N bacteria was grown in a heavy medium and produced a DNA band father down the centrifuge tube. Afterwards, both mediums were mixed together to form a generation and resulted in a DNA band being positioned in the middle of the heavy and light bands.

Ruling out the conservative model but not the other two. In order to figure out which of the two was the replication model, a 2nd generation was generated and resulted in two distinct DNA bands in the centrifuge tube, a light band and a heavy band, ruling out the dispersive model and leading to the semiconservative model being the replication model

What occurs once DNA is unwound?

Two replication forks are formed where DNA is synthesized and move away from each other as replication proceeds

What occurs as incoming nucleoside triphosphates bind to the growing daughter strand?

2 phosphate groups are released from the nucleoside, producing a pyrophosphate, that is then hydrolyzed into 2 separate phosphates and produces energy in the process, providing energy to drive DNA replication

What are the two separate sites present in DNA polymerase?

The polymerizing site for synthesis of new daughter strands and the editing site for the removal of an incorrect nucleotide

How are the 2 sites in DNA polymerase involved in DNA replication?

When DNA polymerase detects an incorrect nucleotide, the newly synthesized DNA strand is unpaired from the template strand and its 3’ end is moved into the editing site, allowing for the incorrect nucleotides to be removed and for the correct nucleotides to be inserted

What do the 5’ and 3’ ends carry that allows for nucleotides to bind?

5’ carries high energy phosphate while 3’ carries low energy hydroxyl

How are RNA primers involved with Okazaki fragments?

They are able to be extended to produce Okazaki fragments with DNA polymerase adding nucleotides to the 3’ end of the RNA primer to synthesize more Okazaki fragments. Once synthesize is completed, RNA primer is removed by nucleases and replaced by DNA

What is present once RNA primers are replaced by DNA?

Gaps called DNA nicks are present between Okazaki fragments

How does DNA ligase join the Okazaki fragments together and fill in the DNA nick?

By hydrolyzing ATP to activate the 5’ phosphate in one fragment and cause it to bind with the 3’ hydroxyl of another fragment, connecting the two fragments

What are clamp loaders?

They are subunit proteins that are responsible for loading DNA polymerase onto the DNA template and ensures it does not fall of the template

What are DNA topoisomerases?

They are proteins that help relieve supercoils by temporarily cutting the strands in order to relieve tension built up in the coils and afterwards DNA ligase binds them back together to continue DNA replication

What is depurination? 

A type of DNA damage that occurs naturally where guanine or adenine are removed from a DNA strand, resulting in a DNA strand without an adenine or guanine base

What is deamination?

A type of DNA damage that removes an amino group from a cytosine, turning it into uracil and resulting in a DNA strand containing a uracil base rather than a cytosine base

How does UV radiation cause external damages to the DNA?

By causing the formation of a dimer between two adjacent thymine’s, resulting in the DNA structure twisting and preventing DNA replication from occurring and introducing errors

What is the affect of deamination in DNA replication?

It can cause the DNA replication machinery to substitute the original base with an alternative base on a new strand when it encounters a uracil on the template strand, causing functional consequences

What is the affect of depurination in DNA replication?

It can cause the DNA replication machinery to delete the incorrect nucleotide position and move on to the next nucleotide pair

• If not deleted then it may be substituted with another base

What occurs in the 3 steps of DNA repair?

1. Excision: the damaged nucleotide is cut by a series of nucleases

2. Resynthesis: Repair DNA polymerase uses the original DNA sequence to restore the DNA strand

3. Ligation: DNA ligase seals the repaired strand in order to reestablish the phosphodiester bond between adjacent nucleotides

What is the DNA mismatch system?

A system that removes any replication errors that escape proofreading during DNA replication

What occurs if an error is left uncorrected?

It can lead to permanent mutations in newly synthesized molecules produced in the next round of DNA replication

How does the DNA mismatch repair system work?

mismatch repair enzymes use the original DNA template to remove any errors from the strand and restore it to its original sequence

What are the two repair methods that occur when DNA is broken in half?

• Non-homologous end joining pathway

• Homologous recombination pathway

With homologous recombination being the better choice and can only happen during DNA replication

What occurs in the Non-homologous end joining pathway?

1. When the break occurs, multiple proteins bind to the break site and recruit repair enzymes to trim the ends of the broken strands

2. The ends are then joined together by DNA ligase and repair is complete but results in some nucleotides being lost in the process

What occurs in the homologous recombination pathway?

1. When the break occurs, nucleases trim off the 5’ ends of the broken strands and leave the 3’ ends alone

2. then the damaged DNA molecule invades an undamaged DNA molecule and repair polymerases uses it as a template to extend the 3’ ends.

3. Once its been completely extended, the 3’ ends will serve as a template for the other ends to fill out the break gap and DNA ligase seals off the gaps