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Flashcards in DNA Replication Deck (18):
1

3 things that DNA replication requires

- ssDNA template
- deoxyribonucleoside triphosphates (dNTPs) as substrates
- DNA polymerase enzyme

2

Describe the ways in which proofreading occurs in DNA replication.

1. BEFORE NUCLEOTIDE IS ADDED: the correct nucleotide will have a higher affinity for DNA polymerase than an incorrect nucleotide
2. 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
3. EXONUCLEOLYTIC PROOFREADING

3

exonucleolytic proofreading

- 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

Why is it that DNA synthesis can only occur 5' to 3'?

- 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

DNA primase (DnaG)

- 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

DNA ligase

- 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

In E.coli, the RNA primer segment is removed by ___ and replaced with dNTPs by ___.

- removed by Ribonuclease H/DNA polymerase I
- replaced with dNTPs by DNA polymerase 𝛿/DNA polymerase I

8

How many nucleotides long are Okazaki fragments in eukaryotes vs. prokaryotes?

- eukaryotes: 100-200 nucleotides long
- prokaryotes: 1000-2000 nucleotides long

9

DNA helicase (DnaB)

- unwinds ("melts") DNA
- hydrolyzes ATP
- propel themselves along ssDNA

10

single stranded binding (SSB) proteins

- 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

β sliding clamp

- 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

strand-directed mismatch repair system (+ MutS, MutL, MutH, DAM methylase)

- 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

DNA topoisomerases

- 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

initiation of DNA replication in bacteria

- 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

Most DNA sequences that can serve as an origin of replication are found to contain...?

- 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

16

chromatin remodeling complexes

destabilize DNA-histone interfaces so that replication fork can advance past parental nucleosomes

17

What are telomeres and what problem do they solve?

- problem with DNA replication in eukaryotes: at the end (after RNA primer removed), there is no 3' OH for DNA polymerase; small amount of DNA would be lost
- telomeres are repeats of short sequences with a block of G nucleotides at the end of eukaryotic chromosomes (e.g. in humans: GGGGTTA)
- protects the ends from nuclease degradation

18

DnaA

- first protein to bind to dsDNA, binds to DnaA-binding region (9bp repeats)
- homotetramer
- causes stress on DNA strands = strand separation