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Biochemistry > Replication > Flashcards

Replication Flashcards

1
Q

Replication

A

Process of DNA synthesis, occurs during S phase and uses DNA polymerase

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2
Q

Protein + nuclear DNA=

A

chromatin

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3
Q

Heterochromatin

A

Very condensed

Contains very few genes. Late replicating and genetically inactive

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4
Q

DNA polymerase needs what to start replicating?

A

Short stretch of RNA serves as a primer

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5
Q

Topoisomerase

A

Relieves overwound supercoils (called DNA gyrase in bacteria-inhibited by the quinolone family of antibiotics)

Breaks a phosphodiester bond

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6
Q

Single-stranded DNA binding protein

A

Binds the single-stranded DNA that has been separated and keeps it exposed in a single-stranded conformation

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7
Q

DNA polymerase α (in complex with primase)

A

Synthesizes RNA-DNA primer

Primase synthesizes an RNA primer that is 7-10 ribonucleotides long and is extended with 25 nucleotides of DNA by DNA polymerase α. The resulting RNA-DNA primer is complementary to a segment of the parental strand

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8
Q

Euchromatin

A

Less condensed

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9
Q

DNA and Histone interaction

A
  • 142 hydrogen bonds formed in each nucleosome
  • Hydrophobic interactions
  • Salt linkages (lysine/argenine are positive and they neutralize negative charge on DNA)
  • Histones are highly conserved
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10
Q

Nucleosome

A

Each nucleosome core particle consists of a complex of 8 histone proteins

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11
Q

DNA polymerase requires what to begin replication?

A

Primer with a free 3’ OH to begin

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12
Q

Sliding clamp

A

Keeps DNA polymerase on DNA when moving; releases when double stranded DNA is encountered

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13
Q

Telomerase

A

Replicates chromosome ends

Special sequence of GGGTTA at end of chromosome

Replenishes these sequences by elongating parental strand in 5’–>3’ direction using an RNA template on the enzyme. DNA polymerase comes in and extends

T-loops: structures protect ends and distinguishes them from broken ones that need to be repaired

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14
Q

Telomere

A

Telomeres are the caps at the end of each strand of DNA that protect our chromosomes, like the plastic tips at the end of shoelaces. Without the coating, shoelaces become frayed until they can no longer do their job, just as without telomeres, DNA strands become damaged and our cells can’t do their job.

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15
Q

Stem cells and telomeres

A

Stem cells retain full telomerase activity

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16
Q

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

Human fibroblasts (synthesizes the extracellular matrix and collagen) normally divide 60 times before undergoing replicating senescence

17
Q

Dyskeratosis congenita disease

A

Carry a mutant telomerase RNA gene

  • Premature shortening of telomeres
  • Die of progressive bone marrow failure
18
Q

We need high fidelity replication rates

A

Germ cells need it to maintain the species

Somatic cells need it to avoid uncontrolled proliferation/cancer

19
Q

What are the two types of spontaneous DNA damage?

A

Depurination and Deamination

20
Q

Depurination

A

The purine base (adenine or guanine) is removed from the nucleotide via hydrolysis of the N-glycosidic bond between the base and the deoxyribose group. This generates an apurinic (AP) or abasic site in the DNA strand

hydrolysis of the N-glycosyl linkage

Repaired beginning with AP endonuclease

21
Q

Deamination

A

The amino group (NH2) of purine or pyrimidine base is hydrolyzed such that adenine is converted to hypoxanthine, guanine is converted to xanthine, and cytosine is converted to uracil, which forms an unnatural deoxyuridine (NH2–> =O)

C–>U

Hydrolysis of the N-glycosyl linkage

22
Q

Methylated cytosines are problematic

A
  • Occurs at some CpG sequences
  • Associated with inactive genes
  • Deamination of methyl-C produces T mismatched with G (NH2–> =O)
  • A special DNA glycosylase recognizes and removes the T
  • Repair is relatively ineffective. Only 3% of C nucleotides are methylated but they account for 1/3 of all point mutations associated with inherited human diseases
23
Q

What does Direct repair (enzymatic repair) fix?

A

Pyrimidine/thymine dimers via DNA photolyase

O6 methylguanine via Methylguanine methyltransferase

24
Q

What does Base excision repair (BER) fix?

A

Single-base mismatches, nondistorting alterations (i.e. depurination)

How?

DNA glycolases (each recognizes a specific type of altered base), AP endonuclease (cut phosphodiester backbone, damage is removed and gap is repaired), AP lyase (of DNA polymerase β), DNA polymerase β, DNA ligase

25
Q

What does Nucleotide excision repair (NER) fix?

A

UV damage (Pyrimidine dimers/thymine)

UV radiation can make a covalent linkage(cross-linking) between two adjacent pyrimidines (T-T or C-T) or thymines

Chemical adducts that distort DNA (i.e. pyrimidine/thymine dimers, BPDE-guanine adducts, cisplatin adducts)

How?

NER protein complex, DNA polymerase Ɛ (epsilon), DNA ligase

26
Q

What disease can be caused by errors in Nucleotide Excision Repair (NER)?

A

Xeroderma pigmentosum

27
Q

What does Mismatch excision repair (MER) fix?

A

Mismatched base in daughter strand

How?

MER complex, helicase/endonuclease, DNA polymerase 𝛿,, DNA ligase

  • MER complex binds to DNA and recognizes mismatch in daughter strand (MutS binds to mismatch, MutL scans for nick and triggers degredation of nicked strand)
  • Daughter strand cut, and segment with mismatch is removed
  • DNA polymerase (delta) fills gap
  • DNA ligase seals nick
28
Q

What disease can be caused by errors in Mismatch excision repair?

A

Hereditary nonpolyposis

Colorectal cancers

29
Q

What does Recombination repair fix?

(Nonhomologous end joining (NHEJ)

Homologous recombination

A

Double-strand breaks, interstrand cross-linking

We have 2 strands so the 1 that is ok can be used as template

30
Q

What happens during Nonhomologous end joining? (part of recombination repair)

A

Damaged ends filled in and joined; some base pairs may be missing. Multiple proteins and enzymes including DNA ligase

You have a break in each of the strands, so you cut out a chunk that encompasses both cuts, and then join the two strands together (you’ll lose some nucleotides b/c of degradation from ends)

31
Q

What happens during homologous recombination? (part of recombination repair)

A

Exonucleases, DNA polymerase, MER system (mismatch excision repair). Damaged duplex repair using information on undamaged homologous duplex

Damage is repaired accurately using info from sister chromatid

32
Q

What disease can be caused by problems in the homologous recombination repair mechanism?

A

BRCA1/2 breast cancer

33
Q

What does Transcription-coupled repair (TCR) fix?

A

Stalled RNA polymerase during transcription (not replication). Directs repair machinery there

34
Q

What disease can be caused by an error in the Transcription-coupled repair (TCR)?

A

Cockayne syndrome

  • Defect in transcription-coupled repair
  • Growth retardation, skeletal abnormalities, sensitivity to sunlight
  • RNA polymerase is permanently stalled at sites of damage in important genes
35
Q

What does Translesion synthesis (bypass synthesis) fix?

A

Unrepaired thymine dimers or apurini AP sites

How?

2 of the reduced-fidelity DNA polymerases (weird greek symbols)

36
Q

Why is RNA not the hereditary information?

A

It cannot distinguish between deaminated Cytosine and natural Uracil

37
Q

How is BER different from NER? (base excision repair vs nucleotide excision repair)

A

NER: Can repair any bulky lesion like those chemically induced and thymine dimers

A multienzyme complex scans DNA for distortion in double helix instead of specific base change

Cleaves phosphodiester backbone on both sides; DNA helicase peels lesion-containing strand away

Large gap is repaired by DNA polymerase and ligase

38
Q

Transcription-coupled repair

A

Cells can preferentially direct DNA repair to sequences that are being actively transcribed by linking RNA polymerase with DNA repair

  • Sequences that urgently need repair
  • Works with BER, NER and others to repair genes that are being expressed when the damage occurs

Biochemistry (41 decks)

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