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Bio 308 > Lecture 6 > Flashcards

Lecture 6 Flashcards

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Biology 101 flashcards
1
Q

Where is DNA replication controlled?

A

At the initiation step

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2
Q

True/False: Both DNA and RNA are negatively charged

A

Ture- which is why binding proteins tend to be positively charged

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3
Q

What is the main difference between replication of origin between prokaryotes and eukaryotes?

A

Prokaryote: Have only one replication origin
Eukaryote: Have multiple origins

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4
Q

Each replication origin have what?

A

Two replication forks, leading and lagging strands, replication bubble

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5
Q

Bidirectionality was proven using what technique?

A

autoradiography- used thymine

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6
Q

How were the enzymes involved in DNA replication discovered?

A

First studied in prokaryotes using both genetic and biochemical approaches

  1. ) Make a mutant population - and screen for mutants that have impaired DNA replication (characterize whether replication stops immediately or slowly under non-permissive conditions)
    2) Purify the enzymes required for replication in invitro
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7
Q

In the method used to identify enzymes in replication, what technique is used to find out about the enzymes?

A

Assay: Where radioactive dNTP are added to an extract containing enzymes necessary for replication, along with template and measured incorporation of radioactive dNTP into new DNA (precipitated and collected on filter)

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8
Q

What are the 10 major enzymes/ proteins we need to know as part of DNA replication?

A

DNA A- INITIATION
DNA B- helicase, DNA C- “clamp loader” , DNA G (primase), RNase H
SSB- single stranded binding proteins
DNA Poly I, III, liagse and Topioisomerase

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9
Q

What is replication mediated by?

A

Replisome: a combination of ALL the proteins that functions at replication fork and undertakes DNA synthesis

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10
Q

What are the three elementary problems of copying DNA?

A

DNA polymerase

  1. ) can’t break down H-bonds
  2. ) can’t start chains, only elongate therefore need primers
    3) can ONLY add at the 3’ OH end
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11
Q

What are the two types of functions involved in the initiation of replication?

A

Cis Elements: cis-acting sites i.e regions and boxes of specific nucleotide sequences on the DNA - CANNOT MOVE AROUND

Trans factors: diffuse through the cell/nucleus. These are proteins that recognize cis elements and bind to them such as DNA A, B and C

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12
Q

True/False: Transcription factors and Trans factors are the same

A

FALSE

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13
Q

What is a key characteristic of origin of replication?

A

Rich in AT - 2 H Bonds

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14
Q

Initiation depends on what at oriC?

A

Methylation by Damm methylase - typically on C and A residues in DNA. N6 of adenine is often targeted

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15
Q

True/False: methylation of DNA happens simultaneously on both strands of DNA?

A

False: Takes time for the newly form strand to be methylated. Thus one strand will have an inactive origin until Dam methylase comes and methylate it

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16
Q

What does dnaA do ?

A

Proteins that initiate replication in E.coli at OriC
Recognize 4 DnaA boxes (9mers) and initiation only occurs is DNA is negatively coiled (easy to melt due to stored energy)
Initial complex= 9mer + 10/20 Dna A proteins

17
Q

How does initial complex turn into open complex?

A

Thorugh the use of ATP (replication bubble in open complex)

18
Q

True/False: Helicases requires ATP to open up the strands

A

True: this group of enzymes needs ATP to move along DOUBLE-STRANDED DNA AND SEPARATE THEM

19
Q

What is helicase known as in E.coli?

A

Dna B

20
Q

What is required to escort Dna B to form a pre-priming complex?

A

Dna C is needed to move DnaB towards Dna A. Dna C also helps to hook the DNA strand into the hexamer (DnaB)

21
Q

What is one quality about DnaB ?

A

It is processive - this means that it will continue until it reaches the end of the strand or it is unloaded by another protein

22
Q

What is the role of single-stranded binding proteins?

A

Cooperative binding: binding of one molecule promote the binding of another
Prevents internal pairing and reformation of double helix

23
Q

Dna G is also known as what?

A

primase aka RNA polymerase to form one RNA primer on leading and several primers on lagging strand

24
Q

What is the Dna B (helicase) + Dna G (Primase) form?

A

Primosome- Primase find helicase to form the next priming site

25
Q

What happens when DNA poly II binds?

A

Initiation is complete and primer is made thus its time for POLYMERIZATION

26
Q

What is the key enzyme in DNA replication in E.coli?

A

DNA POLY III - catalyze synthesis of DNA from Rna primers, forms Okazaki fragments

27
Q

True/False: DNA POLY III composed of 10 different polynucleotides?

A

TRUE: Alpha- for nucleotide addition in 5’-3’, epsilon unit -exonuclease activity of 3’-45’ proofreading, beta subunit used for a clamp,- increase the processivity
Tau protein clams om both leading lagging strand, this is important in ensuring the both single-stranded DNA are replicated at the same time

28
Q

What contributes to the high fidelity of DNA synthesis??

A

3’-5’ exonuclease proofreading activity

29
Q

Can one piece of RNA primer remain on the newly synthesized strand?

A

NO- otherwise will form kink and hinder packaging of the DBA

30
Q

What is the role of RNase H?

A

Auxiliary role: Removes most of the RNA primers except the last one (which is directly linked to DNA)

31
Q

Apart of RNase H, What is another protein capable of removing RNA primers?

A

DNA POLY 1 (specifically its small subunit)- which also have two other abilities: The bigger subunit (Klenow fragment) 5’ to 3’ polymerase replaces the removed RNA primers with dNTP + 3’-5’ proofreading ability

32
Q

Which protein. DNA poly I or DNA POLY III is responsible for the actual DNA strand synthesis?

A

DNA poly III

33
Q

How are the Okazaki fragments joined together?

A

Phosphodiester bonds joining 3’OH ends with 5’ phosphate end of adjacent nucleotides

34
Q

What is the role of topoisomerase?

A

Recognize and regulate supercoiling and involved in DNA replication and transcription

35
Q

What are the two different types of topoisomerase?

A

Topoisomerase I and II

36
Q

Which type of topoisomerase has the biggest role in replication?

A 
Topoisomerase II (gyrase) requies ATP. It introduces negative supercoils around OriC for initiation by Dna A. 
Convert the [positive supercoil formed by helicase as it moves forward to negative supercoils

Bio 308 (6 decks)

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