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BMS-I > DNA Structure/function/replication/repair > Flashcards

DNA Structure/function/replication/repair Flashcards

1
Q

Which nucleic acids bases are purines? Pyrimidines?

A

Purines: A + G
Pyrimidines: C T U

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2
Q

What is a nucleoside?

A

Nucleic acid base with a 5 carbon sugar attached via a N’glycosidic bond

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3
Q

What is a nucleotide?

A

Nucleoside + phosphate via a phosphoesterase bond

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4
Q

CLINICAL EXAMPLE:

How does salmonella cause food poisoning?

A

Inactivation of DNA adenine methylase

  • blocks expression of virulence genes
  • prevent diz development in mice
  • induces an immune response
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5
Q

Clinical correlation:

How are nucleoside analogs used to tx Anti-viral and anti-cancer txs? Give an example:

A

Incorporates into DNA to prevent chain elongation.

Acyclovir - deoxyguanosine analog (HSV)

Azidothymidine (AZT) - analog of deoxythymidine (zidovudive- HIV)

Zalcitabine (dideoxycytidine) - analog of deoxycytidine (HIV)

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6
Q

How to nucleotides polymerize?

A

3’-5’ phosphodiester bonds btwn 3’-OH on sugar of one nucleotide and 5’-P on the next

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7
Q

What is chain polarity of the structure?

A

Free 5’-P at the 5’ end and free 3-OH at 3’end

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8
Q

What is the 1’ sttucture of DNA? 2’? 3’?

A

Primary - nucleotide chain
Secondary - Double helix
Tertiary - supercoiling

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9
Q

What are nucleases? What kind of nucleases do we have?

A

Nucleases - hydrolyze phosphodiester bonds

Exonuclease - cut at an end of a polynucleotide chain

Endonuclease - cleave at internal phosphodiester bonds (i.e. Restriction enzymes, site specifc cleavage)

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10
Q

A restriction enzyme is an example of what kind of nuclease?

A

Endonuclease

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11
Q

What is the most common DNA structure? What is it composed of?

A

B-form
2 anti-parallel polynucleotide chains
5’-3’ & 3’-5’

Right handed double helix

Outer sugar phosphate back bone

Bases perpendicular to axis

10BP/helical turn

Complete turn Q 34A

Chains held together with hydrogen bonds

Minor (narrow) and Major (wide) grooves

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12
Q

CLINICAL CORRELATION:

How do anti-cx drugs DACTINOMYCIN, actinomycin D exert cytotoxic effects?

A

Intercalate at MINOR grooves

Thus interfering with DNA/RNA syn

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13
Q

How does DNA size differ between E.coli and Humans?

A

Ecoli: 1 molecule, circular, DS, 4X10^6bp, 2mm

Human: 46 chromosomes, linear, DS, 6X10^9 BP, 6 geet long

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14
Q

Does a negatively supercoilded DNA double helix have fewer or greater helical turns compared to a relaxed B DNA double helix?

A

Fewer helical turns

I.e. 100BP->10 helical turns
100 -> 8 turns (neg. supercoiled)
100BP->12 turns (pos. Supercoiled)

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15
Q

Why are negative supercoils so important?

A
  1. Facilitate DNA strand separation in double helix (for replication/transcription/repair/recombination)
  2. Energetically favored
  3. NRG needed for strand separation are stored in the supercoils.
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16
Q

Do negative supercoils break phosphodiester bonds?

A

No.

double strand helix is partially unwound (bubbles) and then normal BP is restored.

When eukaryotic histone proteins bind to double helix, histones force DNA to wrap around them . Generates neg supercoil.

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17
Q

What do DNA topoisomerases do?

A

Change tertiary structure (supercoiling)

Swivel points in DNA helix

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18
Q

How does strand separation influence positive supercoiling?

A

Ex. If we have 100BP and uncoil 50, the remainder of 50 BP will be forced to have more helical turns —> (+ supercoiling)

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19
Q

How to topoisomerases work?

A

They have both nuclease and ligase activity.

  1. Transiently break one or both of the DNA strands
  2. Pass the strands thru the break.
  3. Rejoin them
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20
Q

How does TopoI differ from TopoII?

A

TopoI -> cuts single strand of helix

Topo II ->cuts both strands of helix. Think of a loop and all that happens in first step one side is being pulled underneath other. TOPOII comes in and cuts so that now one part of strand loops over and then other loops behind. Religates and now we have a negative supercoil.

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21
Q

What is bacterial DNA gyrase?

A

An unusual TopoII

Can remove both (+ & -) supercoils.

Facilitates bacterial DNA replication

REQUIRES ATP!!!

Introduces negative supercoils into relaxed circular DNA

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22
Q

CLINICAL CORRELATION:

How do drugs targeting Topoisomerases differ from ABX to ACX?

A

ABX -> FQs. Target DNA gyrase (topoII) not found in eukaryotes. Shouldnt have cell cycle specific side effects b/c of this.
Novobiocin - block ATP-binding to gyrase
(Nalidixic acid/Ciprofloxacin - interferes with endonuclease activity of gyrase

ACX ->

Camptothecin -> TopoI

Adriamycin/Etopside ->TopoII
(Converts Topo into DNA breaking agents)

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23
Q

How do prokaryotes and eukaryotes differ in chromatin and chromosome structure?

A

Pro: DNA associated with non-histone proteins. Condense DNA to form nucleoid (nonmembrane bound region)

Eukary: Associated with histone and non-histone proteins
Condensed in the nucleus. Nucleoprotein complex is called chromatin

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24
Q

What are histones?

A

Small BASIC Proteins. Typically ARG and LYS rich

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25
Q

What are the 5 classes of histones?

A

H1, H2A, H2B, H3, H4

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26
Q

What is the structure of the nucleosome?

A

A. Core: Histone octamer w/ DNA supercoiled around (2 histones of h2a/b,h3, h4)
**140BP dna (1&3/4 superhelical turn) winds around histones

B. DNA spacer = 20-80 bp btwn cores, binds with H1.

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27
Q

Why is H1 important?

A

As soon as DNA replication is completed, 1 H1 binds spacer DNA and promotes packing of nucleosomes

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28
Q

What is a solenoid?

A

Helical tubular coil of chromatin

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29
Q

How is compaction of the eukaryotic chromosome completed?

A

Solenoid loops itself and forms large DNA loops (600A) w/ each loop contained 40,000-80,000 BP

DNA loops around scaffold protein.

DNA loops radiate from scaffold = metaphase chromosome (classic 4 arm structure)

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30
Q

When does replication take place in the cell?

A

S phase.

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31
Q

Is replication a conservative process?

A

No, semi-conservative.

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32
Q

How is replication initiated?

A

Partial opening of double helix beginning at origin of replication

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33
Q

What base pairs do we typically see at origins or replication?

A

A:T base pairing

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34
Q

What recognizes AT rich areas of initiation? Is this dependent on energy?

A

DnaA -> induces melting

NEEDS ATP!

35
Q

How many replication forks are created at origin?

A

2

36
Q

What is the role of DnaB in replication? Does it need ATP to function?

A

DnaB (DNA helicase) unwinds the double helix.

ATP dependent yes

37
Q

What are SSBBP role in replication?

A

Single-strand DNA-binding proteins keep DNA single strands apart & protect from nucleases

38
Q

Where is TopoII in relation to the replication fork during replication?

A

Works ahead to remove positive supercoils.

39
Q

CLINICAL Correlation:

How do HSV helicase-primase inhib work?

A

Inhibit progression of HSV DNA replication.

Effective against resistant HSV strains

40
Q

Why are RNA primers important to DNA replication?

A

DNA polymerase cannot recognize ssDNA strands. (Unlike RNA pol)

41
Q

What enzyme adds primers to DNA?

A

Primase (RNA pol) about 10nt

42
Q

What is the polarity of RNA primers?

A

Run in the 5’->3’ direction. Primers provide a free-3’OH as an acceptor of 1st deoxyribonucleotide

43
Q

What strand has the most RNA primers>

A

Lagging strand (Okazaki frags)

44
Q

What is the polarity of the complementary strand of DNA being synthesized?

A

5’->3’ remember DNA is antiparallel

45
Q

What catalyzes chain elongation in prokaryotes?

A

DNA pol III

Nucleophilic attack of 3’-0H terminus on the innermost 5’posphate

46
Q

How is DNA replication regulatd?

A

Proofreading!

There is removal of erroneously introduced nucleotides that are not complementary to the template

47
Q

What type of exonuclease activity does DNA pol III have?

A

3’-5’ (looks behind it)

48
Q

CLINICAL CORRELATION:

What are some examples of drugs that incorporate nucleotide analogs to prevent chain elongation?

A

Acyclovir -> deoxyguanosine (HSV)

AZT -> thymidine analog (HIV)

DdC -> cytidine. (HIV)

Gemcitabine -> Deoxyguanosine (Cancer

49
Q

How are RNA primers removed from strands?

A

5’-3’ exonuclease activity of DNA POL1 removes RNA primers from Okazaki frags.

DNA synthesis cont. with 5’-3’ polymerase activity of DNA Pol I

DNA POL III synthsizes DNA until blocked by primer.

50
Q

What kind of exonuclease activity does DNA pol I have?

A

3’-5’

51
Q

What covalently binds Okazaki fragments? IS this ATP dependent?

A

DNA Ligase - Yes!

DNA Pol III responsible for 5’-P and DNA Pol I responsible for 3’-OH (from removed RNA primer)

52
Q

What are some key differences between Eukaryotic and Prokaryotic DNA replication?

A
  1. More dna
  2. Polymerases and proteins involved
  3. Dna associated with histones
  4. Presence of telomeres
53
Q

What is the role of DNA Pol alpha? Any exonuclease activity?

A
  • DNA replication thru primer synthesis (primase).
  • Initiates syn. On leading and lagging DNA

No exonuclease activity
Primers get removed anyway

54
Q

Role of Pol Beta?

A

DNA repair (Base excision repair -BER)

No exonuclease activity

55
Q

Role of Pol gamma?

A

Mitochondrial DNA replication

3’ -> 5’ exonuclease activity

56
Q

Role of Pol delta?

A

Lagging strand DNA replication
Associated with PCNA to elongate lagging strand
3’-5’ exonuclease activity (looks behind)

Dissociates 5’ end of primers fro Okazaki frag -> degraded by FEN1

DNA repair (MMR, NER)

57
Q

Role of Pol sigma?

A

DNA replication @ leading strand

3’-5’ exonuclease activity

DNA repair (MMR, NER)

58
Q

What are telomeres? How can we recognize them?

A

Ends of linear chromosome

Short non-coding G-rich DNA repeat (TTAGGG)

Form t-loops

59
Q

What are the roles of telomeres?

A

A.Protecting ends of linear chromosomes

  1. Recognition as broken DNA and subsequent degradation.
  2. Recombination
  3. End-to-end fusion

B. Prevent loss of important coding terminal sequences.
Since primers are needed for DNa replication

60
Q

What is the “end problem” that telomeres solve?

A

Primer removal would result in loss of DNA information. This adds info to 3’ end of strand so primer can bind on and initate lagging strand length

61
Q

How does telomerase work?

A

Ribonucleoprotein.

Adds G-rich DNA repeats to SS 3’ends of linear chromosomes.

Protein component has REVERSE transcriptase activity.

62
Q

When is telomerase most active?

A

Stem/Germ b4 birth. Telomeres shorten with each cell division

63
Q

What happens when chromosome length declines to a critical point?

A

End-to-end fusions (no more t-loops)

64
Q

What is the role of p53 in relation to telomeres?

A

P53 is a dna damage sensors. Should initiate cell growth arrest

In cancers, telomerase may be reactivated with loss of p53.

65
Q

CLINICAL correlation:

What is Dyskeratosis congenita?

A

Inheritied disease with reduced telomerase activity. (Mutations in gene that encodes for RNA component of telomerase)

Affects highly proliferative tissues

Alopecia, gut disprders, lung fibrosis

66
Q

What is difference betwen Dyskeratosis congenita and Hutchinson-Gilford progeria?

A

HG progeria = accelerated telomere shortening
(alopecia, aged skin appearance, die from MI

Dyskeratosis = reduced telomerase activity (die from BM failure)

67
Q

What pathway would replication errors be fixed through?

A

MMR (single extra nucleotide/mismatch)

68
Q

How would DNA repair damage to a single base?

A

BER (deamination/depurination/alkylation/oxidation)

69
Q

What are the ways DNA could repair bulky DNA adducts

A
  1. Global genomic- NER
  2. Transcription coupled NER

(Induced via radiation/chemicals, distortion of helix)

70
Q

How are SSB incorporated into DNA?

A

Oxidative damage

71
Q

How are DS DNA breaks incorporated into DNA? How would we fix these?

A 
Ionizing radiation (gamma/xrays)
Oxidizing agents (bleomycin)
Topo inhib (camptothecin ->topoI

Fixed via:
1. Non-homologous end joining

  1. Homologous recombination
72
Q

What enzymes are involved in MMR pathway?

Proteins? How do we recognize?

A
  1. Endonuclease
  2. Exonuclease
  3. Helicase
  4. Polymerase (POL III / delta/sigma +PCNA)
  5. Ligase

Proteins:

E.coli - MutS/MutL
Human - MSH2/6 (mismatch)
MSH2/3 (1-4 nt insertions/deletions)

73
Q

CLINICAL CORRELATION:

What cancers are lynch pts mor elikely to have? What do 90% of these pts have?

A

Colorectal (+endometrial/skin/ovarian/gastric/renal)

MSH2 or MLH1

74
Q

What enzymes are involved in the BER pathway?

A
  1. Glycosylase
    (recognizes damaged base), cleaves n-glycosidic bond)
  2. Endonuclease (apurinic/apyrimidic cleaves sugar backbone)
  3. Deoxyribose P lyase (removes sugar-phosphate residue)
  4. Polymerase (DNA POL 1 (Beta) fills in new base
  5. Ligase
75
Q

CLINICAL Correlation:

How do defects in BER manifest?

A

A. Mutation in gene encoding for MYH glycosylase.
Increased risk for colon cancer.

B. Mutations in RecQ DNA helicase WRN

Werner’s syndorme: Rare autosomal recessive disorder
WRN helicase involved with BER

76
Q

CLINICAL CORRELATION:
UV damage induces thymine dimers & smoking has carcinogens. What pathway corrects this DNA damage? What mutations would these cause if uncorrected?

A

Nucleotide excision repair (NER)

Frameshift

77
Q

What enzymes are involved in NER? Whats the difference between Global genomic and TC?

A
  1. Helicase
  2. Excinuclease
  3. Polymerase
  4. Ligase

Only mechanism that removes bulky adducts.
GG-NER => transcriptionally inactive region
TC-NER => active region

78
Q

What pathogensis does GG-NER defect lead to? TC-NER? Why? Can it be both?

A

GG-NER: cancer w/o CNS disorder
TC-NER: CNS w/o cancer (blocks progression of RNA pol along transcribed gene)

GG-NER = Xeroderma Pigmentosum (XP)
-mutations in XPC/XPE/XPD/XPA, affecting damage recognition activity. Cancers increased, can be part of common pathway which would also lead to neuro deficits.

TC-NER = Cockayne syndrome (CS)
Hereditary developmental and neurological disorder associated mutations in CSA/CSB (affect recognition of stalled RNA pol II)
Growth and mental retardation

79
Q

Why is cisplatin useful for NER deficient tumor cells?

A

MOA: forms bulky intra-strand adducts with DNA.

80
Q

What enzymes are involved in SSB repair?

A

Recognition by PARP1 & recruitment of XRCC1 to serve as scaffold to recruit multiple repair proteins. Ex. Aprataxin APTX

  1. Hydrolase/transferase
  2. Polymerase
  3. Ligase
81
Q

What diz is deficient SSB repair associated with?

A

Ataxia Oculomotor Apraxia (AOA1)
-autosomal recessive spinocerebellar ataxia
Uncoordinated gait, peripheral neuropathy,

Resembles Ataxia telangiectasia (AT) w/o immunodeficient featues

Caused by mutation in APTX gene

82
Q

What enzymes are needed for DSB repair? Proteins? Recognition?

A
  1. NHEJ - major pathway, mutagenic
    A. Broken DNA sensor
    (Ku70/80 binds DNA ends, recruits DNAPKcs-Artemis)
    B. Nucleases (artemis has exonuclease activity)
    C. Polymerases
    D. Ligases
  2. Homologous recomination - minor, non-mutagenic
    Restricted to S/G2 phases
    A. Recombinases (RAD52 binds DNA ends, rad51 looks for homology (mediated by BRCA1/2)
    B. Endonucleases
83
Q

What is Ataxia telangiectasia?

A

Autosomal recessive with mutation in ATM protein (that is normally activated by DSBs)

Increased chromosomal abnormalities (t+B cells)
Develop lymphoid cancer.

84
Q

How does a mutation in an allele of BRCA1/2 affect lifetime risk of developing cancer?`

A

Increases by 80%

BMS-I (17 decks)

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