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Molecular Biology - Unit 1 - Zach H. > Lecture 3: DNA Replication, Repair, and Recombination 1 > Flashcards

Lecture 3: DNA Replication, Repair, and Recombination 1 Flashcards

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1
Q

What is the germ-line error rate (mutation rate) in humans?

A
  • About 70 new single-nucleotide mutations in the offspring’s germline when compared to parental germline.
  • Mutation rate of one nucleotide change per 1x10^8 nucleotides per generation.
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2
Q

How are most germ-line errors corrected?

A
  • MOST errors are corrected by:
    • proofreading (polymerase activity)
    • DNA repair
  • Errors are further corrected by post-replication repair mechanisms.
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3
Q

Why is it so crucial for multicellular organisms to have high fidelity replication?

A
  • Germ cells have to have low mutation rates to maintain the species.
  • Somatic cells need low mutation rates to avoid uncontrolled proliferation/cancer.
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4
Q

What is the catalyzing reaction by DNA polymerase when synthesizing DNA?

A

(DNA)n+1 residues + dNTP -> (DNA)n+1 + P2074-

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5
Q

What does DNA polymerase require to begin?

A

Requires a primer with a free 3’-OH to begin.

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6
Q

What is released from the reaction when DNA polymerase addes a new deoxyribonucleoside triphosphate to the growing strand during replication?

A

Pyrophosphate

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7
Q

True or False:

DNA polymerase can only synthesize DNA in the 5’-to-3’ direction.

A

True

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8
Q

True or False:

The replication fork is symmetric.

A

False - replication fork is asymmetric.

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9
Q

Between the leading and lagging strand, which one is synthesized continuously and which one is synthesized in segments?

A
  • Leading Strand = synthesized continuously
  • Lagging Strand = synthesized in segments
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10
Q

What fragment is produced in segments during lagging strand synthesis?

A

Okazaki Fragments

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11
Q

How often does DNA polymerase make a mistake?

A

Every 1x109 nucleotides copied.

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12
Q

How are the mistakes that DNA polmerase make sensed by the replication machinery and fixed?

A
  • Before a new nucleotide is added the enzyme must tighten its “fingers” around the active site, which is easier if the correct base is in place.
    • If incorrect base - this is sensed by the palm not being able to close down correctly, which trigger the proofreading (exonuclease) activity of DNA polymerase.
  • DNA polymerase has exonuclease activity in the 3’-to-5’ direction that will fix the incorrect base.
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13
Q

When does exonucleolytic proofreading take place?

A

Immediately after incorrect base is added.

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14
Q

During exonucleolytic proofreading, what does the exonuclease do if the base is incorrect?

A
  • 3’-to-5’ exonuclease clips off unpaired residues at 3’ primer terminus.
    • DNA polymerase requires a perfectly paired 3’ terminus to continue replication.
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15
Q

Why is it important that replication proceeds in the 5’-to-3’ direction?

A
  • 5’-to-3’ replication allows efficient error correction.
    • in a hypothetical 3’-to-5’ strand growth, if proofreading takes off a nucleotide then the reaction does not proceed, as no high-energy bond would be cleaved (thus, not favored by the cell).
    • in 5’-to-3’ strand growth, if proofreading takes off a nucleotide then a high-energy bond is cleaved, providing the energy for polymerization (thus, favored by cell).
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16
Q

What type of process is the lagging strand replicated through?

A
  • Backstitching Process:
    • DNA primase synthesizes an 10 nt long RNA primer to prime DNA synthesis.
      • DNA polymerase can’t initiate de novo synthesis - this would increase the mutation rate.
    • RNA primer is erased by RNAseH (recognizes RNA/DNA hybrids) and replaced with DNA; DNA ligase joins the ends.
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17
Q

You know that inorder to prime DNA synthesis DNA primase must synthesize a 10 nt long RNA primer. What enzyme erases the RNA primer?

A

RNAseH (recognizes RNA/DNA hybrid) and replaces it with DNA; DNA ligase joins the ends.

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18
Q

At what rate can DNA helicase unwind DNA?

A

Capable of prying apart the helix at rates of 1000 nucleotides pairs/sec.

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19
Q

How many subunits make up DNA helicase?

A
  • Protein with 6 identical subunits that binds and hydrolyzes ATP.
    • this causes conformational change that propels it like a rotary engine along single stranded DNA, passing it through a center hole.
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20
Q

What is the function of single-stranded DNA binding proteins (SSB)?

A
  • Bind tightly and cooperatively to exposed SS DNA.
    • help stabilize unwound DNA
    • prevent formation of hairpins
    • DNA bases remain exposed

**bind to keep the single stranded DNA so the DNA polymerase can stay on and correctly replicate long stretches**

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21
Q

What is the function of the sliding clamp?

A
  • Keeps DNA polymerase on DNA when moving.
    • releases when double stranded DNA is encountered
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22
Q

What is the function of the clamp loader?

(hint: assembly requires clamp loader)

A

Hydrolyzes ATP as it loads the clamp onto a primer-template junction.

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23
Q

True or False:

On the leading strand the clamp remains associated to DNA polymerase for long stretches.

A

True

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24
Q

True or False:

On the lagging strand the clamp loader stays close so it can assemble a new clamp at the start of each new Okazaki fragment.

A

True

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25
Q

What does mismatch repair do?

A

Removes (almost all) errors missed by proofreading by detecting distortion caused by mispairing.

26
Q

How does mismatch repair know which strand is correct?

A
  • In E. coli, depends on methylation to distinguish new strand.
    • MutS binds to mismatch
    • MutL scans for the nick and triggers degradation of nicked strand
  • In humans, depends on single strand breaks.
    • present on lagging strand before Okazaki fragments are ligated.
    • leading strand not known
27
Q

What happens if there is mutations in the mismatch repair gene?

A
  • cells accumulate mutations at high rate
  • HNPCC
    • Hereditary Nonpolyposis Colorectal Cancer
28
Q

Look over slide 17 - 2015 Nobel Prize in Chemistry

A

Given to three sicentists for “mechanistic studies of DNA repair”.

29
Q

What is the function of DNA topoisomerases?

A
  • As replication fork moves, it creates a winding problem for the parental helix.
  • Every 10 bp replicated corresponds to one turn.
  • DNA topoisomerase is a reversible enzyme that breaks a phosphodiester bond to change superhelicity, thereby relieving supercoiling.
30
Q

What is the function of type I topoisomerase?

A
  • Catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process.
  • Work by creating transient single strand break in DNA which allows the DNA on either side of the nick to rotate freely relative to each other.
    • uses the other phosphodiester bond as a swivel point
  • Resealing is rapid and doesn’t require any addition energy since energy is stored in the phosphotyrosine linkage.
31
Q

What function does type II topoisomerase have?

A
  • Type II enzymes make a transient double-stranded break in the DNA.
  • Activated at sites on chromosome where two double-stranded helices cross each other:
  • It uses ATP to:
    • 1) break one double-stranded helix reversibly to create “gate”
    • 2) causes second strand to pass through
    • 3) reseals break and dissociates
  • It can separate “decatenate” 2 interlocked DNA circles
  • Can prevent severe tangling problems that would arise during DNA replication.
32
Q

What are the 3 things that type II topoisomerase uses ATP to do?

A
  1. Break one double-stranded helix reversibly to create “gate”.
  2. Causes second strand to pass through.
  3. Reseals break and dissociates.
33
Q

What nucleotides make up the nucleotide rich sequence where the replication origin is found?

A

A-T rich regions where sequence attracts initiator proteins to pry open DNA.

34
Q

True or False:

Initiation is the only point of control for E. coli, so it’s highly regulated.

A

True

*proceeds only when sufficient nutrients are present

*refractory perior - delay until new strand is methylated

35
Q

Why is it enough for bacteri to have only 2 replication forks, but not eukaryotes?

A
  • These two replication forks are enough for bacterial genomes, which are small.
    • only 40 minutes to replicate entire genome
  • Eukaryotes need more
    • traveling at 50 nt/sec, it would take 800 hours to do an average chromosome with a single ORI.
36
Q

What is the general mechanism of initiation of DNA replication in bacteria?

A
  • Initiator proteins bind to specific sites in ORI, forming complex.
  • This complex attracts DNA helicase + helicase loader
  • Helicase is placed around a ssDNA exposed by assembly of the complex.
  • Helicase loader remains engaged until helicase is properly loaded.
  • Helicase unwinds DNA so primase can make RNA primer on leading strand.
    • remaining proteins assemble to create 2 replication forks with complexes moving in opposite direction with respect to the ORI.
37
Q

What phase of the cell cycle does DNA synthesis occur? Also, about how long does it lasts in mammalian cells?

A
  • Occurs during DNA synthesis phase (S).
  • About 8 hours for mammalian cells.
38
Q

True or False:

Chromosomes are replicated to produce two complete copies, joined at centromeres until M phase in the cell cycle.

A

True

39
Q

You know that replication is activated in clusters (replication units). But how many replication origins are located in each cluster?

A

consists of 20-80 replication origins

40
Q

Is heterochromatin late- or early replicating during S phase?

A

Late-replicating

  • Example: X chromosomes of females:
    • almost all of inactive X is condensed into heterochromatin and is replicated late in S phase.
      • the active homolog is less condensed and replicates throughout S phase.
41
Q

What are the three minimum requirements for a sequence to be an ORI in yeast?

A
  1. must have binding site for ORC (origin recognition complex).
  2. must have an A-T rich stretch for easy unwinding.
  3. must have binding site for proteins that help attract ORC.
42
Q

What proteins bind to form the pre-replication complex and regulate origin activity?

A
  • Helicase
  • Helicase loading proteins:
    • Cdc6
    • Cdt1
43
Q

In the S phase of the cell cycle, what do activated Cdk proteins lead to?

A
  • dissociation of helicase loading proteins
  • activation of helicase
  • unwinding of DNA
  • loading of DNA polymerase, etc.
44
Q

What control mechanism prevents assembly of a new ORC until the next M phase?

A

Once the pre-replication complex is formed, the ORC is phosphorylated to prevent it from replicating a second round before the cell divides. Once the cell divides, then the ORC will be de-phosphorylated to activate it again.

  • single chance to form in G1 when Cdk activity is low.
  • second window for pre-replicative complexes to be activated and disassembled in S phase when Cdk’s activity is high.
45
Q

True or False:

ORI function depends critically on distant sequences.

A

True - if there is DNA deletions in either the ORI or the thousands of nucleotide pair distant sequences, then the indiated origin of replication is inactive.

  • Specific human sequences have been identified that can serve as ORIs; they are 1000s of nucleotide pairs in length.
  • They can still function if moved to a different locus if placed where chromatin is uncondensed.
  • Example: distant DNA in beta-globin cluster is required for expression of the genes in the cluster.
    • ORI function depends critically on distant sequences.
      • also affects transcription
      • global effect of decondensing chromatin structure.
46
Q

For efficient replication, what is needed to destabilize the DNA-histone interface?

A

Chromatin remodeling proteins.

47
Q

As the replication fork passes through chromatin, what does the histone octamer break into? Also, which dimers are found on the daughter nucleosomes?

A
  • Breaks into:
    • an H3-H4 tetramer, distributed randomly to daughter duplexes
    • 2 H2A-H2B dimers which are released from the DNA
48
Q

Why are histone chaperone proteins needed during reassembly?

A
  • Freshly made H3-H4 fills in spaces; H2A/H2B dimers are half old and half new; they are added at random to complete complex.
  • Directed to DNA with sliding clamp called PCNA.
49
Q

What are the two histone chaperone proteins that we covered in class?

A
  • NAP-1
    • loads H2A-H3B dimer
  • CAF-1
    • loads newly synthesized H3-H4 tetramer
50
Q

True or False:

Some daughter nucleosomes contain only parental histones or only new ones, but most are hybrids of old and new.

A

True

51
Q

Patterns of histone modification can be inherited. Howare these histone modifications spread?

A

By the reader-writer complexes.

*may be responsible for some types of epigenetic inheritance*

52
Q

What is the special sequence that is repeated about 1000 times at the end of each chromosome?

A

GGGTTA

53
Q

Why is there a end replication problem on the lagging strand? How is this overcame?

A
  • Because there is not a place for an RNA primer.
  • Thus, an enzyme called telomerase replenishes these sequences by elongating parental strand in 5’-to-3’ direction using an RNA template on the enzyme.

*Bacteria have circular genomes so they don’t have telomers like eukaryotes have*

54
Q

What happens after the parental strand has been extended by telomerase?

A

Replication of the lagging strand can be completed by DNA polymerase, using the extension as a template.

  • this mechanism (plus a 5’ nuclease) ensures that the 3’ end is longer, leaving a protruding SS end that loops back and tucks into the repeat (called T-loop).
55
Q

Why are T-loops important at the end of chromosomes?

A
  • Structures protect ends and distinguishes them from broken ones that need to be repaired.
  • Shelterin (protein) - protective chromosome cape made up of proteins.
56
Q

Do stem cells retain full telomerase activity?

A

Yes

57
Q

True or False:

Human somatic cells are born with full complement of telomere repeats.

A

True

58
Q

Why are telomere repeats lost after each generation?

A

Due to insufficient telomerase activity.

59
Q

What is replicative senescence?

A

After many generations, daughter cells will have defective chromosomes and stop dividing; in this way the cell’s lifetime is regulated to guard against cancer.

60
Q

How many times do human fibroblasts normally divide before undergoing replicative senescence?

A

60 times.

**continue dividing when telomerase is provided experimentally**

61
Q

What disease in humans is characteristic of the patient carrying a mutant telomerase RNA gene?

A
  • Dyskeratosis Congenita
    • develop prematurely shortened telomeres
    • die of progressive bone marrow failure
62
Q
A

Molecular Biology - Unit 1 - Zach H. (5 decks)

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