DNA Replication Flashcards
1
Q
3 things that DNA replication requires
A
- ssDNA template
- deoxyribonucleoside triphosphates (dNTPs) as substrates
- DNA polymerase enzyme
2
Q
Describe the ways in which proofreading occurs in DNA replication.
A
- BEFORE NUCLEOTIDE IS ADDED: the correct nucleotide will have a higher affinity for DNA polymerase than an incorrect nucleotide
- BEFORE NUCLEOTIDE IS COVALENTLY ADDED TO GROWING CHAIN: DNA polymerase must undergo a conformational change to increase grip around the base pair; conformational change occurs more readily with the correct nucleotide
- EXONUCLEOLYTIC PROOFREADING
3
Q
exonucleolytic proofreading
A
- incorrect nucleotide may be added but not base paired
- if nucleotides are not base paired, DNA polymerase cannot continue elongation
- 3’-to-5’ proofreading exonuclease is a separate catalytic site on a separate domain or subunit of polymerase; will clip off incorrect residue
4
Q
Why is it that DNA synthesis can only occur 5’ to 3’?
A
- this is the only way a mismatched base can be corrected
- the 3’ end allows for high energy phosphate bonds to be cleaved so an incoming dNTP can be added and polymerization can occur
- if 3’ to 5’, after the removal of a mismatched base there will be no source of high energy phosphate bonds to be cleaved so polymerization terminates
5
Q
DNA primase (DnaG)
A
- uses ribonucleoside triphosphates to synthesize short RNA primers
- ~10 nucleotides long
- only one needed on the leading strand
- on the lagging strand, one primer is added every 100-200 nucleotide intervals
- forms DNA/RNA hybrid
6
Q
DNA ligase
A
- joins 3’ end of new DNA fragment to the 5’ end of the old one with ATP
- req. to make lagging strand a continuous strand of DNA
7
Q
In E.coli, the RNA primer segment is removed by ___ and replaced with dNTPs by ___.
A
- removed by Ribonuclease H/DNA polymerase I
- replaced with dNTPs by DNA polymerase 𝛿/DNA polymerase I
8
Q
How many nucleotides long are Okazaki fragments in eukaryotes vs. prokaryotes?
A
- eukaryotes: 100-200 nucleotides long
- prokaryotes: 1000-2000 nucleotides long
9
Q
DNA helicase (DnaB)
A
- unwinds (“melts”) DNA
- hydrolyzes ATP
- propel themselves along ssDNA
10
Q
single stranded binding (SSB) proteins
A
- helix destabilizing proteins
- work together to expose ssDNA
- help helicase by stabilizing, straightening and preventing reannealing of ssDNA
- prevents formation of hairpin helices of ssDNA that would block DNA polymerase
11
Q
β sliding clamp
A
- keeps DNA polymerase firmly attached to ssDNA until it reaches dsDNA when it releases it (since DNA pol. has a tendency to rapidly dissociate)
12
Q
strand-directed mismatch repair system (+ MutS, MutL, MutH, DAM methylase)
A
- detects potential for distortion of DNA helix from misfit or non complementary base pairs
- MutS dimer recognizes mismatch
- MutH binds to daughter strand
- MutL dimer binds to MutS and activates SHL complex
- after these mismatch proofreading proteins binds, DNA scanning detects nick in the new DNA strand
- strand (between error and nick) is removed and repaired by DNA pol. and ligase
13
Q
DNA topoisomerases
A
- relieve the overwinding of DNA in front of the replication fork
- reversible nuclease covalently adds itself to phosphate of DNA backbone, cleaving the phosphodiester bond (which is reformed when the nuclease leaves)
- single strand break (topoisomerase I) or double strand break (topoisomerase II)
14
Q
initiation of DNA replication in bacteria
A
- initiator proteins bind to replication origin and destabilize A-T rich sequence
- loading of DNA helicase (DnaB) bound to helicase-loading proteins
- helicase activated when helicase-loading proteins dissociate
- loading of DNA primase (DnaG), synthesizes RNA primers
- RNA primers allow DNA polymerase to start new chains (leading strand synthesis begins)
- 2 additional DNA polymerases loaded (lagging strand synthesis begins)
15
Q
Most DNA sequences that can serve as an origin of replication are found to contain…?
A
- binding site for a large initiatior protein, an origin recognition complex (ORC)
- stretch of DNA rich in As and Ts that is easy to melt
- at least one binding site for proteins that facilitate the binding of ORC
