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Molecular Bio - Exam I > Lect. 3 - Replication, repair and recombination 1 > Flashcards

Lect. 3 - Replication, repair and recombination 1 Flashcards

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Biology 101 flashcards
1
Q

mutation rate in human germ line

A

one nucleotide change per 10^8 nucleotides per generation

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2
Q

mutation rate in e.coli

A

one nucleotide change per 10^10 nucleotides per cell generation

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3
Q

What is a critical need for multicellular organisms?

A

HIGH FIDELITY REPLICATION; Germ cells have to have low mutation rates to maintain the species and somatic cells need low mutation rate to avoid uncontrolled proliferation/cancer

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4
Q

DNA polymerase

A

synthesizes DNA by catalyzing the following rxn: (DNA)n residues + dNTP –> (DNA)n+1 residues + P2O7

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5
Q

What does DNA polymerase require to begin?

A

a primer w/ a free 3’ -OH to begin

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6
Q

What direction can the DNA polymerase synthesize in?

A

5’ to 3’ direction

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7
Q

How often does DNA polymerase make a mistake?

A

1 out of every 10^9 nucleotides copied, thanks to proofreading.

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8
Q

What step is first just before a new nucleotide is added?

A

enzyme must tighten its “fingers” around the active site, which is easier if the correct base is in place

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9
Q

Exonucleolytic proofreading

A

DNA polymerase requires a perfectly paired 3’ terminus’ 3’ to 5’ exonucleuase clips off unpaired residues at 3’ primer terminus

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10
Q

When does exonucleolytic proofreading take place?

A

immediately after incorrect bases is added

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11
Q

Why 5’ to 3’?

A

Allows for efficient error correction; that direction is energetically favorable to remove a new chain and start over.

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12
Q

Lagging strand synthesis

A

replicated through backstitching process

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13
Q

what does DNA primase do?

A

synthesizes an 10nt long RNA primer to prime DNA synthesis b/c if DNA polymerase initiated it it would increase mutation rate

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14
Q

RNA primer

A

erased by RNAseH (recognizes RNA/DNA hybrids) and replaced w/ DNA;

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15
Q

What joins the ends after DNA replaces the RNA?

A

DNA ligase

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16
Q

DNA helicase

A

unwinds DNA; protein w/ 6 identical subunits that binds and hydrolyzes ATP; this confomational change that propels it like a rotary engine along single stranded DNA, passing it through a center hole.
Capable of prying apart the helix at rates of 1000 nucleotide pairs/sec

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17
Q

Single-stranded DNA binding proteins

A

bind tightly and cooperatively to exposed SS DNA; it is less energetically favorable so will want to try and make a double strand if possible (or bind to itself = hairpin)

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18
Q

what 3 things do the single-stranded DNA binding proteins do?

A
  1. help stabilize unwound DNA
  2. prevent formation of hairpins
  3. DNA bases remain exposed
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19
Q

Sliding clamp

A

keeps DNA polymerase on DNA when moving; releases when double stranded DNA is encountered (allows long stretches of DNA rep. to occur)

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20
Q

clamp loader

A

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

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21
Q

On the leading strand, what does the clamp do?

A

remains associated to DNA polymerase for long stretches

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22
Q

On the lagging strand, what does the clamp loader do?

A

stays close so it can assemble a new clamp at start of each new Okazaki fragment

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23
Q

Mismatch Repair

A

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

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24
Q

Which strand is correct?

A

the methylated strand is correct in e.coli and in humans, depends on single strand breaks - present on lagging strand before Okazaki fragments are ligated, leading strand not known.

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25
Q

MutS

A

binds to mismatch

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26
Q

MutL

A

scans for the nick and triggers degradation nicked strand

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27
Q

DNA Topoisomerases

A

reversible enzyme that breaks a phosphodiester bond to change superhelicity, thereby relieving supercoiling

28
Q

what makes up one turn in DNA?

A

every 10 bp replicated

29
Q

Type 1 Topoisomerases

A

Catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process.

30
Q

How do Type 1 Topoisomberases work?

A

by creating a 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

31
Q

What is the resealing like in type I Isomerases?

A

rapid and doesn’t require any additional energy since energy is stored in the phosphotyrosine linkage

32
Q

Type II Topoisomerase

A

make a transient double-stranded break in the DNA

33
Q

What do type II topoisomerase use 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
34
Q

decatenate

A

2 interlocked DNA circles that can be separated by type II topoisomerase

35
Q

Where/when are type II topoisomerases activated?

A

at sites on chromosomes where 2 double-stranded helices cross each other

36
Q

Replication origins

A

A-T rich (only 2 H bonds) regions where sequence attracts initiator proteins to pry open DNA

37
Q

What is the only point of control for E. coli?

A

initiation - that is why it’s highly regulated

38
Q

Regulation of initiation in e. coli

A

proceeds only when sufficient nutrients are present; refractory period - delay until new strand is methylated

39
Q

initiation of DNA replication in bacteria

A

initiator proteins bind to specific sites in ORI, forming complex which attracts DNA helicase + helicase loader

40
Q

where is helicase places?

A

around a SS DNA exposed by assembly of complex

41
Q

Function of helicase

A

unwinds DNA so primase can make RNA primer on leading strand; remaining proteins assemble to create 2 replication forks w/ complexes moving in opposite direction w/ respect to the ORI

42
Q

In Eukaryotic DNA Replication, when does it occur and how long does it last?

A

occurs during DNA synthesis phase (S) which lasts about 8 hrs for mammalian cells (so will have a lot of different origins of replication)

43
Q

in eukaryotic DNA replication, what replicates first?

A

regions of genome w/ less condensed chromatin replicate first

44
Q

in yeast (proks), what are the 3 minimum requirements for sequence to be ORI?

A
  1. must have binding site or ORC (origin recognition complex)
  2. Must have an AT rich stretch for easy unwinding
  3. Must have binding site for proteins that help attract ORC
45
Q

what are the helicase loading proteins involved in regulation of proks

A

Cdc6 and Cdt1

46
Q

In S phase, what does activated Cdks lead to?

A

dissociation of helicase loading proteins, activation of helicase, unwinding of DNA, and loading of DNA polymerase, etc.

47
Q

Prevention of assembly of new ORC

A

prevent assembly of new ORC until next M phase resets cycle; single chance to form in G1 when Cdk activity is low;

48
Q

When is the second chance for pre-replicative complexes to be activated

A

can be activated and disassembled in S phase when Cdks activity is high

49
Q

What is an important part for mammalian ORIs?

A

Chromatin Structure

50
Q

if ORIs are moved to a different locus can they still function?

A

Yes, as long as they are placed where chromatin is uncondensed

51
Q

replication requires not only DNA replication but what else?

A

synthesis and assembly of new proteins

52
Q

when are histone proteins mainly synthesized?

A

S phase; amount made is highly regulated to meet requirements

53
Q

As replication fork passes through chromatin, histone octamer breaks into what?

A
  • and H3-H4 tetramer, distributed RANDOMLY to daughter duplexes
  • 2 H2A-H2B dimers which are released from the DNA
54
Q

What does reassembly require?

A

Histone chaperones

55
Q

What is the make up of H2A/H2B?

A

they are half old and half new - they are added at random to complete complex

56
Q

What is the sliding clamp called that directs the chaperones (chromatin assembly factors)?

A

PCNA

57
Q

Patterns of histone modification can be inherited

A

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

58
Q

What replicates the chromosome ends?

A

telomerase

59
Q

Why is end replication a problem on the lagging strand?

A

because there is no place for RNA primer - not a problem for bacteria b/c they have circular genomes

60
Q

What special sequence is at the end of each chromosome?

A

GGGTTA - repeated about 1000x

61
Q

Telomerase

A

enzyme that replenishes these sequences by elongating parental strand in 5’ to 3’ direction using an RNA template on the enzyme

62
Q

Telomerase Replication

A

after extension of parental strand by telomerase, replication of lagging strand can be completed by DNA polymerase, using extension as template

63
Q

What does the telomere replication mechanism ensure?

A

that the 3’ end is longer, leaving a protruding SS end that loops back and tucks into the repeat

64
Q

T loops

A

structures protect ends and distinguishes them from broken ones that need to be repaired

65
Q

Shelterin

A

protective chromosome cap made up of proteins

66
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

67
Q

What may be responsible for aging in animals?

A

replicative senescence

Molecular Bio - Exam I (9 decks)

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