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chapter 5 Flashcards

1
Q

DNA replication

A

A-T and G-C, each daughter same as parent

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2
Q

AT and GC how many bonds

A

AT- 2H (easier to break)

GC- 3 H bonds

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3
Q

ORC

A

is where replication takes place

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4
Q

DNA replication steps

A
  1. Initiator proteins binds to origin (AAA, ATP) (helices bind and unwind DNA)
  2. DNA strands separated at AT rich
  3. Initiator protein recruits replication proteins

– ATP (regulates) by Orc1 needed for Orc to bind to DNA

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5
Q

Initiation: bacteria E.coli

A

origin OriC has a 245bp sequence

STEPS:
1. bound to ATP, AAA+ domains of DnaA = make a spiral filament

  1. DnaA also recruits DnaB to the origin
  2. DnaB is a helicase, loaded onto the DNA by DnaC

A- unwinds (initiator)
B- recruit’s
C- opens up

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6
Q

Initiation: Eukaryotes

A

Origin is ~100bp long

  1. A1 and B1 sites that the ORC binds to AT-rich sites are B2 and B3- downstream
  2. ORC recruits Cdc6 and Cdt1, recruit the MCM helicase proteins
  3. make the prereplicative complex
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7
Q

Initiation: Single-stranded Binding Proteins

A

can form secondary structures that would make copying by polymerase difficult

SSB in bacteria
RPA in eukaryotes

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8
Q

Initiation: Helicases

A

must be in single not dd to replicate

STEPS:
1. Open up the helix and continuously unwinding it at the replication fork as it moves

  1. Bacterial helicase moves 5-3
  2. Eukaryotic helicase moves 3-5
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9
Q

Helicase, DnaB: Pro

A

binds ssDNA and is displaced

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10
Q

Steps for Eu: Helicases

A
  1. DNA helicase unwinds helix to expose ssDNA
  2. ssDNA coated with ssDNA binding proteins
  3. DNA synthesis needs a primer

Add nucleotides only to an existing 3’end
Primer is a short RNA strand synthesized by primase

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11
Q

Elongation

A
  1. loading sliding clam and loader
  2. loader of replicate polymerase
  3. fork movement (5-3)
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12
Q

Sliding clamp

A
  • ring shape 35A
  • keeps DNA polymerase to DNA
  • Binds DNA polymerase through an 8 AA
  • stable
Bacteria= B protein
Eukaryotes= PCNA
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13
Q

what is a clamp loader

A
  • 5-subunit ring structure
  • Replication Factor C (RFC) in eukaryotes
  • Some are ATPases

ATP-> change -> clamp loading -> spiral shape

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14
Q

clamp loader steps

A
  1. ATP binding
  2. 3 end of primer enters CL
  3. ATP hydrolysis, clamp closes + released
  4. clamp gets DNA pol
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15
Q

Type II: topoisomerase

A

STEPS:

  1. Both strands of DNA are cut and bound to tyrosine residues
  2. dsDNA segment passes through gap
  3. DNA re-ligated with some supercoiling relieved
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16
Q

Termination occurs when?

A
  1. two different forks meet
  2. a fork reaches the end of a linear chromosome
  3. polymerase meets previously replicated strand
17
Q

Termination- Eukaryotes

A
  • linear chromo

- complex= histones

18
Q

Termination- prokaryotes

A
  • circular chromsome
  • less complex
  • @ter site
  • ter bound to tus= disassembly

makes type I1 and II topo

19
Q

both termination- prokaryotes and Eu have

A
  • three phases of replication
  • Replication catalyzed by DNA polymerases
  • Require helicases, single-strand binding proteins, primases, initiator proteins, topoisomerases, and ligases
20
Q

DNA polymerases types

A

DNA polymerase III in bacteria

DNA polymerase signa and E in eukaryotes

21
Q

DNA polymerase: structure

A

Thumb: holds elongated dsDNA
Fingers: positions incoming dNTP
Palm: connects all domains

22
Q

DNA polymerase: active site

A

carboxylate groups (2 aspartates) coordinated with 2 magnesium

Catalyzes a phosphoryl transfer reaction by Nucleophilic attack by the
• 3ʹ-OH on the α-phosphate of incoming nucleotide

Mg: One activates the 3-OH
One interacts with dNTP to stabilize the negative leaving oxygen

23
Q

DNA polymerase: Fidelity

A
  1. has precise fit in active site when base-paired with the template strand
  2. Mismatches = different shape and don’t fit in the active site as well
  3. No energy is required

•Incorrect dNTPs nevertheless sometimes added to DNA strand

24
Q

Proofreading

A
  1. Polymerase moves on to next dNTP, but slows when an incorrect addition was made
  2. Exonuclease removes incorrect nucleotide
  3. Energy is required
  4. 3ʹ end of strand returned to polymerase active site
25
Q

Reverse transcriptase

A

DNA polymerases that copy ssRNA into DNA

Encoded by viruses and by DNA elements in eukaryotes called retrotransposons

26
Q

Chromatin Replication

A
  1. Asf1 and Caf1 bind and with the sliding clamp then deposit new H3 and H4 onto DNA behind replication fork
  2. Newly synthesized H2A and H2B complete the nucleosome
  3. H3 and H4 are then deacetylated-
27
Q

The End-Replication Problem

A

Lagging strand synthesis
Cannot replicate very end of linear chromosome

  1. RNA primer removal - Leaves gap
  2. dissolution of the replication fork
28
Q

The End-Replication: solve

A

telomeres!

Simple sequence repeats on chromosome ends

TTAGGG repeats in human G-rich strand

Ends of telomeres get shorter after each round of replication

29
Q

Telomerase

A

DNA polymerase made of protein and RNA molecule

RNA template used to synthesize DNA-repeated many times from RNA template (TERT) conserved in eukaryotes

Binds to single-stranded telomere DNA (G-rich strand)

3ʹ end of the DNA base-pairs with the telomerase RNA

30
Q

Regulation of Initiation: (E. coli)

A
  1. DnaA binds at oriC when bound to ATP
  2. After initiation, Hda & sliding clamp stimulates hydrolysis of the DnaA-ATP- unwinds DNA for replication initation
  3. After initation, stimulates hydrolysis of the DNAA-ATP
  4. DnaA-ADP dissociates

This Regulatory Inactivation of DnaA (RIDA) blocks replication initiation

DARS regions- reactitive dnaA-ATP

31
Q

Regulatory Inactivation of DnaA (RIDA)

A

blocks replication initiation

32
Q

DARS regions

A

reactitive dnaA-ATP

33
Q

Regulation of Initiation: (E. coli): After chromosome replication

A
  1. DnaA binds the datA DNA region

2. After replication, two datA regions= more DnaA bound and less available to bind oriC

34
Q

Regulation of Initiation: (E. coli)After segregation of chromosomes

A

One datA again= dnaA free to bind oriC and promote initation

35
Q

Regulation of Initiation: Eukaryotes: origins

A
  • Selected in G1
  • Activated in S
  • ORC is at the center of a larger complex
  • (pre-RC) forms at the origin in G1, only be assembled in G1 and includes Cdt1, Cdc6 and MCM helicase
36
Q

Regulation of Initiation: Eukaryotes: steps

A
  1. pre-RC is phosphorylated by S phase Cdks, making it active
  2. origins to unwind, and activates the MCM helicase complex
  3. After origin firing - complex inactivated, cannot re trigger initiation until next cycle
  4. Replication must be completed before chromosome segregation occurs

advanced (8 decks)

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