Lecture 4 - DNA Replication, Recombination, & Repair

Question 1

Requirements for DNA Replication

Answer 1

• Template - provides sequence information
• Primer - provides free 3’ -OH to which nucleotides are added
• Precursors - 5’ deoxynucleoside triphosphates (5’-dNTPs)
• Enzymes - DNA polymerase, sliding clamps, helicases, primases, single-stranded DNA-binding proteins, nucleases, ligases

Question 2

DNA polymerase (type & activity)

Answer 2

• Pol alpha/primase - priming and synthesis of initial part of Okazaki fragment
• Pol delta - synthesis of leading strand & most of Okazaki fragment, gap filling after removal of primer

Question 3

Helicase

Answer 3

Burns ATP and splits parental strands causing replication fork to progress

Question 4

Single-stranded DNA-binding proteins

Answer 4

Needed to keep parental strands from reannealing after separation by helicase

Question 5

DNA ligase

Answer 5

Seals pieces of Okazaki fragments together (final bond that DNA polymerase can’t make)

Question 6

Topoisomerase

Answer 6

• makes nicks (in front of helicase) in one of the DNA strands to relieve torsional constraint and then seals nick after strain is relieved
• target for antibiotics (ciprofloxacin)
• cipro causes topoisomerase to remain bound to DNA to block replication

Question 7

Clamp-loading protein

Answer 7

Binds to DNA and assembles sliding clamp

Question 8

Sliding clamp

Answer 8

associates with DNA polymerase and allows it to remain attached to the DNA

(think of sliding clamp like a washer)

Question 9

Key eukaryotic proteins involved in replication

Answer 9

• RPA (SSBs)
• Pol alpha/primase
• PCNA/RCF (PCNA = sliding clamp)(RCF = clamp loading protein)
• Pol. delta
• Rnase H/Fen1 (nucleases) - digest primers
• DNA ligase

Question 10

End-replication problem

Answer 10

Daughter strand will be shorter than parental strand because there is no place to synthesize a primer that would allow the daughter strand to be completed

Question 11

Telomerase

Answer 11

• Functions as a reverse transcriptase and extends the parental telomere
• After this extension, the daughter strand can be primed and extended
• Cancer cells have overactive telomerase which keeps cell alive too long

Question 12

Types of mutations

Answer 12

• Point mutations
• Insertions and deletions
• Triplet expansion

Question 13

Basic steps of excision repair of DNA

Answer 13

Recognition of damage
Remove damage
Resynthesize to fill gaps
Ligate to restore continuity

Question 14

Xeroderma Pigmentosum

Answer 14

• Inability to repair thymine dimers
• 1st disease demonstrated to be caused by defective DNA repair
• Pts are photosensitive and highly susceptible to skin cancers (2k-5k fold higher than avg.)
• Many pts also have neurological problems
• rare autosomal recessive disease (any of 8 different genes)

Question 15

Types of DNA repair

Answer 15

• Excision repair
• Mismatch repair
• Translesion DNA synthesis - special DNA pol. can instert nucleotides opposite the lesion allowing replication to proceed
• Double-stranded break repair - uses Ku and DNA-PK