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BIO282 > DNA Replication > Flashcards

DNA Replication Flashcards

1
Q

Significance of DNA replication

A
  • Is a fundamental process
  • Of interest, as things do go wrong during it
  • Viral replication can be useful to understand as it can be a potential target
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2
Q

Model of DNA replication

A

-Is found to be semiconservative (new products have 1 newly syn. strand and 1 old strand)

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3
Q

Replication of DNA in bacteria

  • why done
  • how it works for E. coli
A

-Bacteria smaller, easier to grow

  • replication begins at Origin of replication; replication forks move bidirectionally around circular chromosome
    • at replication fork, there is a complex of proteins -> are responsible for carrying out replication
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4
Q

Replication of Eukaryotic DNA

-enzyme responsible

A
  • Similar concepts to prokaryotic
  • Eukaryotes differ in that they have >1 origin of replication w/ forks moving away from this origin
  • bidirectional replication holds true for all cellular systems
  • DNA polymerase responsible for synthesising DNA
    • DNA poly 1 first identified (is the one that synthesises DNA)
    • adds to 3’ end of the primer
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5
Q

DNA polymerase

  • what it needs, direction of synthesis
  • How lagging strand is synthesised (and by what)
A
  • Needs a primer to add bases to
    • synthesises in a 5’ to 3’ direction
  • 2nd strand is synthesised as a series of fragments (called Okazaki Fragments) that are subsequently drawn together
  • DNA polymerase adds to RNA primers that are synthesised by primase (is an enzyme part of complex at replication fork)
    • DNA polymerase 3 responsible for extending RNA primers (fills in the spaces)
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6
Q

RNA primers in DNA replication

  • how they’re synthesized
  • How they are removed - what happens
A
  • Are produced by de novo synthesis -> is very error prone (NOT GOOD)
    • are replaced with DNA
  • After DNA poly 3 fills in gaps between primers, it dissociates
  • DNA poly 1 binds to the bap between the DNA and RNA primer
    • DNA poly 1 has a 5’ to 3’ exonuclease that allows it to degrade DNA or RNA in front of it - fills in behind it
      • this action removes RNA and replaces it with DNA
  • gap is sealed by DNA ligase
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7
Q

Processivity

A
  • Is the number of bases added to a primer before the enzymes disassociates
  • Differs between types of polymerases
  • DNA poly 1 has processivity of 15-20 (about the length of RNA primer)
  • DNA poly 3 holds DNA in place - is a very big subunit
  • DNA poly 1 is a single monopeptide unit
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8
Q

Separating the DNA strands

  • why need to separate
  • how can be done
A
  • need to separate the strands to replicate
  • can be achieved by supercoiling -> usu allows the DNA to fit inside the cell
    • super coiling with right twist = condense
    • super coiling with left twist = separation of DNA strands
  • can have molecules that are half condense and half relaxed (are enzymes that can reach the same effect)
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9
Q

Topoisomers -> what they are and what they do

Helicases -> also what they do

A

*Enzymes that introduce supercoils or relax supercoils in DNA

Helicases: enzymes that disrupt the H bonds that hold the two strands of the double helix together (responsible for ‘unzipping’ the DNA molecule)

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10
Q

DNAa protein

  • what it does and effect it causes
  • another factor that enables it do its job more easily

-what happens once job is done

A
  • is a critical protein in DNA replication
  • Binds to specific seq. at origin of replication
    • DNA monomers start binding to each other and cause bending and twisting of DNA at that position
      • Denaturation occurs at this site
  • This site is also AT rich = more easily denatured (2 H bonds instead of 3)
  • Once area denatured, other proteins come in (i.e. DNAb protein) and drives the process of DNA replication
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11
Q

DNAa protein, DNAb protein, DNAc protein

  • > what they do and how they interact
  • DNA polymerase 3 - what is it’s role?
  • DNAg Primase
A
  • DNAa protein binds to specific sequence (DnaA box - are 5 at origin) that unwinds the origin
    - additional DnaA proteins bind to newly unwound single-stranded regions
  • DNAb protein drives the process of DNA replication (are helicases that unwind DNA)
    - are 2 monomers of DNAb (one at each replication fork)
  • DNAc protein helps DNAb bind to DNA
  • DNA polymerase 3 = major enzyme to replicate DNA (need monomers/complexes - one for each strand)
  • DNAg primase = synthesises RNA primers
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12
Q

Replisome assembly -> overview

-DnaA’s job

A
  • Assembly of replisome = orderly process that begins at precise sites on chromosome called Origins
  • after unwinding begins after the binding of DnaA proteins, additional DnaA proteins bind to single stranded region
  • 2 helicases now bind and slide in a 5’ to 3’ position to begin unzipping helix at replication fork
  • Primase and DNA polymerase 3 holoenzyme recruited to replication fork by protein-protein interactions & DNA synthesis begins

*DnaA job = bring the replisome to the correct place in the chromosome for initiating replication

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13
Q

Components of Replisome (6)

-what is not included

A
  • DNAb
  • DNA polymerase 3
  • DNAg primase
  • DNA polymerase 1
  • DNA ligase (seals gaps in strand)
  • Single strand binding (SSB) protein

*DNAa is not included because it just aligns replisome to right spot

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14
Q

Role of SSB protein - why is it needed

A
  • As DNA/RNA doesn’t like to be single stranded, it will H bond with itself
  • In region of replication fork where there is s.s. DNA, it will form loops (cruciform loops)
    - in replication, DNA polymerase tends to miss these structures are therefore hotspots for mutations
  • SSB binds to DNA & keeps it in extended form for replication and prevents intrastrand base pairing
    - decreases mutations/lost DNA
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15
Q

The replisome - functions of parts;-

clamp proteins
helicase
DNAg primase
SSB proteins

*what happens to rest of DNA molecule in front of helicase??

A
  • Clamp proteins: hold DNA polymerase in place on DNA
  • helicase unwinds strands
  • DNAg primase synthesises small RNA primers
  • SSB proteins binds to s.s. DNA

*rest of DNA molecule gets knotted up infront of helicase -> is another topoisomerase that unknots the DNA before the helicase gets to it

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16
Q

Movement of replisome

-term for end of ‘cycle’

A
  • Replisome moves around the circular chromosome until reaches point opposite the origin = TERMINUS
    • at this point, the linked circles are separated by a topoisomerase
  • the separated circles then migrate to opposite poles of the cell
17
Q

Replication in Eukaryotes -> how it is different

How to get rid of RNA primers and replace it with DNA? (Overview)

A
  • Eukaryotes have linear molecules
  • multiple ORI
  • If RNA left, will get degraded in cell
  • to get rid of RNA primer, can use 3’ end of chromosome (are repeats of a short sequence motif = telomeres)
    • may be 10^5 to 10^7 telomeres added to the chromosome
      • in human somatic cells, they are not replaced (gets shorter after every round of division (other species, telomerase replaces these repeats)
18
Q

What structures allow RNA primers to be replaced with DNA

  • what these structures provide
  • what telomeres do -> an absence of telomeres
A
  • Hairpin structures allow RNA primers to be replaced w/ DNA
    • telomeres at each end of chromosome (they also protect chromosome from degradation by nuclases)
  • Hairpin loop provides 3’ end where DNA polymerase can bind and replace RNA primer

-Progeria: Where people age very quickly due to having very short telomeres

19
Q

Another way to replicate the ends of the chromosome

-and what organism usually uses this

A

-Use protein primers
-DNA polymerase is able to bind to cytosine and initiate synthesis of the complementary strand
(Cytosine is attached to a protein that binds to the template)
*these are mainly used in viruses

20
Q

Why is DNA replication linked to cell division?

A
  • Replication of DNA followed by cell division
    • each daughter cell needs a copy of the chromosome
  • variation in chromosome number is not acceptable
  • process of cell division must be linked to the process of DNA replication
21
Q

Cell cycle (4 phases)

A

G1 -> S -> G2 -> M
G1: interphase (cell growth) - need enough cell components for cell division to occur and support 2 daughter cells
S phase: DNA replication
G2: Recovery from DNA replication - is where the cell checks the newly replicated DNA for mistakes (as replication occurs v. quickly & genomes can be huge)
-if too many mistakes, cell undergoes apoptosis
M phase: where mitosis occurs

22
Q

Result of having same cell cycle operating in each cell

A

-Each daughter cell has a copy of the genome

23
Q

Cell cycle in bacteria (Overview)

A
  • bacteria are roughly similar, but less complex (don’t have spindle fibres, intracellular membrane systems etc)
    • therefore divide by binary fission
  • they grow, replicate dna and then segment DNA for 2 cells (chromosomes attach to membrane of cell - when new membrane grows at centre of cell, it pushes cell’s genome apart)
24
Q

Regulation of the cell cycle

  • how it is usually balanced
  • what unregulation leads to
    • what we have learnt from this
A
  • Eukaryotic cells do not divide continuously - process is regulated so that organs are the correct size
  • cell division & apoptosis is in constant state of equilibrium
  • Unregulated cell division = cancer
    • studying cancer cells have led to the identification of many cell cycle regulatory proteins
25
Q

Oncoviruses - what they are

-how they work

A
  • Viruses that cause cancer
  • Do so by infecting cell, integrating into the genome of the cell and conferring ability of uncontrolled replication to the cell
    • been found that oncoviruses carry altered versions of normal genes
26
Q

DNA replication and regulatory proteins

-cycle of regulatory proteins in cell

  • MPF - what it is
    • when it is most concentrated in cell

-what it does

A

-process of DNA replication & cell division are tightly regulated -> regulatory proteins wouldn’t be present at all stages of cell cycle (would only be present when required & would show a cyclical pattern)

MPF = Mitosis promoting factor -> key regulatory protein that induces cell entry into M phase

  • accumulates to its highest level at M phase of the cell cycle
  • stimulates condensation of chromosome, spindle formation and breakdowns nuclear membrane
  • it is a cyclin dependent kinase
27
Q

MPF -> cyclin dependent kinase segment

-what each part does

A
  • Cyclin = selects target protein
  • Kinase = phosphorylates proteins
  • works in G2 to M
  • cyclin degraded after M phase (is what determines specificity) - kinase can be reused
  • is a similar regulatory process (with kinase) from G1 to S phase
28
Q

Regulatory system of Cell division in bacteria

-How regulatory protein (what is it?) is controlled

A
  • Is similar to eukaryotic
  • DNAa protein is the critical protein -> it accumulates in cyclic fashion too (peak = active form of protein)
  • When bound to ADP = inactive
    • or can bind to high affinity sites away from OriC (bind it to something else so it can’t initiate new rounds of replication)
      • i.e. seqA protein (therefore can’t bind to OriC)
29
Q

How can bacteria grow so quickly?

A
  • Bacteria can initiate new rounds of replication before other rounds are completed
    • eukaryotes can NOT do this

BIO282 (17 decks)

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