Lecture 3: DNA repair, and homologous recombination

Question 1

how many errors per nucleotide added between each combined. replication step

Answer 1

1 in 10^10 nt copied

Question 2

what are the 3 most common dna damages

Answer 2

• depurination
• depyrimidination
• cytosine deamination

Question 3

what are different kinds of spontaneous nucleotide modifications

Answer 3

• oxidative damage
• hydrolytic attack like depurination, depyrimidaytion, and C deamination
• methylation

Question 4

true/false without DNA repair, spontaneous DNA damage would rapidly change DNA sequences

Answer 4

true

Question 5

what does depurination do to a dna strand

Answer 5

• the N‐glycosidic bonds are cleaved to release the corresponding adenine or guanine from DNA
• the purines

Question 6

what does deamination do to a dna strand

Answer 6

• converts cytosine to uracil
• can occur on other bases as well
• removes amines, and adds oxygen

Question 7

what base is formed if adenine is deaminated

Answer 7

hypoxanthine

Question 8

what base is formed if guanine is deaminated

Answer 8

xanthine

Question 9

what base is formed if cytosine is deaminated

Answer 9

uracil

Question 10

what base is formed if thymine is deaminated

Answer 10

thymine cannot be deaminated (no amine group)

Question 11

what is a thymine dimer

Answer 11

a photolesion produced by UV radiation in sunlight and is considered as a potential factor causing skin cancer

Question 12

how is a thymine dimer formed

Answer 12

formed as a covalently bonded complex of two adjacent thymines on a single strand of DNA

Question 13

what does uracil DNA glycosylase do

Answer 13

removed deaminated cytosine

Question 14

describe what happens when a 5-methyl C nucleotide is deaminated

Answer 14

• about 3% of the C nucleotides are methylated to help control gene expression
• when they are accidentally deaminated, they form the natural T
• this T will be paired w a G, making a mismatched base pair

Question 15

describe how base excision repair works

Answer 15

• uracil dna glycosylase removed the deaminated cytosine
• AP endonuclease recognizes a deoxyribose sugar with a missing base (the gap thats formed from removing the cytosine)
• the sugar phosphate with a missing base is cut out by sequential action of AP endonuclease and a phosphodiesterase
• the gap of a single nucleotide is then filled and sealed by DNA polymerase and DNA ligase

Question 16

describe how nucleotide excision repair works

Answer 16

• a multi-enzyme complex recognizes a lesion such as a pyrimidine dimer
• a cut is made on each side of the lesion
• a DNA helicase then removes the entire portion of the damaged strand
• DNA polymerase and DNA ligase fills in the missing DNA

Question 17

what process is nucleotide excision repair coupled to and why

Answer 17

• transcription
• to ensure fidelity

Question 18

when are translesion DNA polymerases used, explain the process

Answer 18

• when there is damage on a dna strand
• this can stall replicative dna polymerases
• covalent modifications will happen to the sliding clamp when the polymerase encounters DNA damage
• the replicative dna polymerase will be released, and the translesion one will load on
• its more versatile and less accurate
• once the damage is bypassed, replicative polymerase will return

Question 19

are double-strand breaks common

Answer 19

yes

Question 20

what are 2 ways to repair a double-strand break

Answer 20

• nonhomologous end joining
• homologous recombination

Question 21

desribe non-homologous end joining

Answer 21

• broken ends are ligated without the need for a homologous template
• typically uses short-homologous DNA sequences (microhomologies) to guide repair, present in single-strand overhands on the ends of double strand breaks
• Ku protein grasps the broken chromosome ends
• additional proteins hold the broken ends together while they are processed and joined together covalently

Question 22

what is a repurcussion of non-homologous end joining

Answer 22

repaired DNA has generally suffered a deletion of nucleotides

Question 23

describe how homologous recombination works

Answer 23

• nuclease digests 5’ ends of broken strands, creating an overhang
• one broken strand attaches the overhang part to the homologous piece of DNA
• repair polymerase synthesizes DNA using undamaged DNA as a template
• the damaged DNA is then released once the replication has happened
• this extended damaged DNA is then used as a template for the other strand of the damaged DNA
• dna ligation will occur

Question 24

when is homologous recombination used

Answer 24

when there is a homologous piece of DNA present in the nucleus (ie during DNA replication time of cell cycle)

Question 25

what happens to a replication fork if it encounters a single-strand break

Answer 25

it will collapse

Question 26

describe how replication forks recover after encountering a single-strand break

Answer 26

• via homolgous recombination
• nucelase degreades 5’ end
• strand exchanges and DNA synthesis happens
• replication fork restarts

Question 27

which deamination is not readily repeared

Answer 27

5-methyl cytosine to thymine