DNA replication Flashcards by LV D

Which types of proteins are involved in DNA synthesis and what is their function?

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What are the main differences btw pro- and eukaryotic DNA replication?

in eukaryotes:

many ORC (instead of oriC) b/c much lower rate of replication → replication bubbles instead of replication fork
SSBs are called replication protein A (RPA)
different DNA polymerases
DNA has to be rearranged into chromosomes after replication

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Compare speed and error rate (fidelity) of DNA replication (in pro-/eukaryotes), RNA transcription and protein translation.

DNA replication: very low error rate (high fidelity) ~ 10^-8- 10^-9, a bit slower in eukaryotes due to nucleosomal interactions
RNA transcription/translation: slower than replication, much higher error rate ~ 10^-4

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Differentiate btw prokaryotic DNA polymerases.

Which ones are involved in DNA replication?

Pol I: DNA replication (RNA primer removal), DNA repair
Pol II: DNA repair
Pol III: DNA replication

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Which factors contribute to the high fidelity of DNA replication in prokaryotes?

intrinsic fidelity of DNA polymerase: 10^-3 - ^-4
sensing dNTP complementarity to template
exonuclease proofreading of DNA polymerase: 10^-2 - ^-3
sensing primer complementarity to template
mismatch repair: 10^-2
sensing complementarity of 2 strands, distinguishing parental/daughter strands

⇒ overall: 10^-8 - 10^-9

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Differentiate btw eukaryotic DNA polymerases.

Which ones are involved in DNA replication?

Pol α: DNA replication (primer synthesis) = primase
Pol β: base excision repair
Pol γ: mitochondrial DNA replication/repair
Pol δ: DNA replication (lagging strand synthesis), nucleotide/base excision repair
Pol ε: DNA replication (leading strand synthesis), nucleotide/base excision repair

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Which reaction is catalyzed by DNA polymerases (in eukaryotes and prokaryotes)?

dNTP + OH- on 3’ end as primer
→ DNA polymer w/ phosphodiester bonds + PPi
BUT: require template strand + Mg²⁺ as cofactor

⇒ synthesizes 3’→5’, daughter strand formed 5’→3’

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How are both DNA strands synthesized?

leading strand synthesized continuously
lagging strand synthesized discontinously, Okazaki fragments are formed (in prokaryotes ~ 1000 bps, in eukaryotes ~ 200 bps)

⇒ bidirectional synthesis in replication bubble

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How is mismatch basepairing recognized by DNA polymerases?

DNA polymerase recognizes differences in atom distances + bond angles

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What is special abt DNA pol I?

coded by pol A: mutants lack proofreading activity
monomer

only prokaryotic DNA pol w/ 3 activities b/c involved in replication and repair

3’→5’ polymerase activity
5’→3’ exonuclease (essential for initial removal of RNA primer)
3’→5’ exonuclease (proofreading)

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How does DNA pol I know when to use its polymerase or proofreading activity?

Klenow segment of DNA pol I has P and E site

when template connected to primer passing through P site: polymerisation
when template unwinds from primer + translocates to pass through E site: editing

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What is the function of DNA pol II?

participates in repair, requires duplex template and primer

coded by pol B: mutation is not lethal
multimeric (4+)

only 3’→5’ exonuclease activity (proofreading)

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Describe the function of the structural elements of DNA pol III.

2 DNA pol III form an asymmetric dimer w/ multiple subunits

each α subunit: 3’→5’ polymerase activity
β subunits: act as sliding clamp, keep enzyme attached to DNA
γ subunit: acts as clamp loader for lagging strand Okazaki fragment, help β subunits to form unit + bind to DNA
each ε subunit: 3’→5’ exonuclease activity (proofreading stimulated by Θ subunit)
τ subunits: dimerize, tether both enzymes together

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What is the function of DNA pol III?

replicates prokaryotic DNA

coded by pol C: mutants are temperature sensitive, conditionally lethal

3’→5’ polymerase activity +
3’→5’ exonuclease activity (proofreading)

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What is the mechanism of 5’→3’ exonuclease activity of DNA pol I?

removes RNA primer on lagging strand + fills in the necessary nucleotides between the Okazaki fragments in 5’→3’ direction, proofreading for mistakes as it goes

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What is the first step of the initiation of DNA replication in prokaryotes?

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initiatior protein DnaA activated by ATP binding, multiple DnaAs bind to AT-rich 4bp regions of oriC (2 H-bonds, easier to unzip)

→ local denaturation + unwinding

What happens during replication in prokaryotes once the dsDNA is partially unwound?

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recruitment of DnaB (5’→3 helicase), binds to lagging strand to start its action at AT-rich 13 bp regions of oriC = DUE (DNA unwinding elements)

→ ATP hydrolysis to unwind DNA to ssDNA

NOTE: binds to DnaG during elongation

The DNA is now “fully” unwound to ssDNA.

What happens now in prokaryotes?
(still part of initiation)

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recruitment of DNA gyrase (topoisomerase I)

→ relieves the torsional stress caused by unwinding the double helix

Describe the mechanism of topoisomerase I

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NOTE: topoisomerase I causes single strand break

Describe the mechanism of topoisomerase II.

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NOTE: topoisomerase II causes double strand break

List therapeutic implications related to topoisomerases.

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antibacterial quinolones (ex: ciprofloxacin) also targets bacterial topoisomerase I (DNA gyrase)
topoisomerase I and II poisons used in chemotherapy
- topo I: topotectan
- topo II: daunorubicin, doxorubicin, etoposide

How is the replication fork kept open after unwinding in prokaryotes?

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SSBs (single-strand binding proteins) bind to lagging strand + stabilize ssDNAs to maintain the replication bubble

Once the two strands are seperated, elongation begins…

What has to happen first though? In prokaryotes.

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DnaG (primase) binds to leading and lagging strand to synthesize RNA primers to the template strands

leading strand receives 1 RNA primer
lagging strand receives several primers
NOTE: binds here to DnaB (helicase) to form primosome

What happens now, after attachment of primers to both DNA strands? In prokaryotes.

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DNA pol III dimer binds w/ β subunits (sliding clamp) to leading strand, w/ γ subunits (clamp loader) to lagging strand

→ synthesizes daughter strands, beginning at 3’-OH of primer/s

leading strand extended continuously
lagging strand extended discontinuously forming Okazaki fragments

NOTE: τ subunits tether both dimers together, so that lagging strand synthesizing DNA pol III doesn’t leave replication bubble

DNA pol III is not the only DNA polymerase involved in the elongation process in prokaryotes. Also another one binds, what does it do?

**DNA pol I** binds to _lagging strand_ → **removes RNA primer + fills gaps** w/ dNTPs

What has to happen to each of the Okazaki fragments in prokaryotes?

**DNA ligase seals** _single nick_ (missing phosphodiester bond btw adjacent dNPTs) on leading strand, seals _multiple nicks_ on lagging strand **mechanism** (see picture) _NOTE:_ uses **NAD⁺**

How is the mechanism of DNA ligase in eukaryotes different from that in prokaryotes?

uses _**AMP** for adenylation_ of DNA ligase instead of NAD⁺

How is DNA replication in prokaryotes terminated?

* either when both replication forks meet * when replication fork reaches **Ter sequences →** either _completely block_ termination or allow replication only to continue in one direction (c_lockwise/counterclockwise fork traps_)

What is ARS and how was it identified in yeast?

**ARS** = _autonomously replicating sequence_ yeast requires His to grow, but if *his gene* missing, His cannot be produced, hence no growth possible 1. in some yeast cells, **plasmids** introduced containing ARS (have oris) 2. although His absent, population w/ ARS was able to independently replicate probably means the same as episome

What are telomeres?

region of _repetitive nucleotide sequences_ at each end of a eukaryotic chromosome that form **T-loops** * protect the ends of the chromosome from **deterioration or from fusion** with neighboring chromosomes * **protect DNA from shortening** _{(each round of replication leaves 50-200 bps unreplicated at 3' end b/c the synthesis of Okazaki fragments requires RNA primers attaching ahead on the lagging strand)}

Describe the structure of the enzyme responsible for formation of telomeres.

**telomerase** = reverse transcriptase w/ RNA template 4 subunits: * _fingers + thumb_ * _palm:_ active site * _remainder of telomerase RNA:_ used as template for binding to DNA and synthesis of telomeres

Describe the mechanism how telomerase synthesizes telomeres.

1. **telomerase binds** w/ RNA template to 3' end of newly synthesized lagging strand 2. **telomerase extends 3' end**, using its RNA-template for complementary base pairing 3. **telomerase relocates**, repeats step 2 4. **DNA polymerase** complements lagging strand

Explain how mtDNA is replicated.

_bidirectional_, but **asynchronous**, starts w/ H strand 1. D loop = origin of replication for H strand 2. RNA primer generated from L strand transcript 3. after 2/3 of H strand replicated, origin of replication for L strand uncovered (on old H strand) when DNA polymerase displaces old H strand 4. L strand origin folds into a stem-loop structure, acting as RNA primer, so that replication starts here

How is the DNA reorganized into chromatin after replication?

newly snyth. and preexisting **histone octomers** are **randomly distributed** among both arms of the replication fork and **reorganized into nucleosomes** facilitated by _chaperones_

What are retroviruses?

viruses that transcribe their **genetic RNA material via reverse transcriptase into DNA** which is eventually incorporated into host DNA → can be mutagenic, inactivate genes or disregulate its gene expression

DNA replication Flashcards

(35 cards)