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Ch. 20
DNA Replication
37
Biochemistry
Undergraduate 3
12/01/2010

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Term
Meselson/Stahl Exeperiment
Definition
Through the use of DNA labeled with the heavy isotope N15, they showed that each of the daughter DNA molecules have one strand of parental DNA and one strand of new DNA, thus DNA replication is SEMICONSERVATIVE.
Term
What direction does DNA Replication go?
Definition

DNA Polymerase can only make DNA in the 5'-->3' direction

Leading strand is continuously replicated in the 5'-->3' direction, the lagging strand is replicated discontinuously as a series of Okazaki Fragments working in opposite direction of replication fork but still being replicated in a 5-->3 direction

Term
From what does DNA Replication start?
Definition

RNA primers formed by PRIMASE primes synthesis

Prokaryotes have a particular site called the ORIGIN

Eurkaryotes do not have a site but rather have a structure that is located by an origin recognition complex

Term
What are the 7 proteins involved in DNA Replication?
Definition

Helicase

SSB's

Primase

DNA Polymerase

Sliding Clamp

RNase H

Ligase

Term
What is Helicase (and what does it do)?
Definition

an enzyme made up of six proteins arranged in a ring shape

 

starts DNA synthesis by unwinding the double helix exposing the 2 single DNA strands

 

requires ATP

Term
What are SSBs and what do they do?
Definition
Single-strand binding proteins- tetramers that coat the single stranded DNA and prevent it from annealing. These are easily displaced by DNA Polymerase
Term
What is primase and what does it do?
Definition

An RNA-polymerase that synthesizes the short RNA primers needed to start the strand replication process

The RNA primer is then extended by DNA polymerase

Term
What is DNA polymerase and what does it do?
Definition

a hand-shaped enzyme that strings nucleotides together to form complementary DNA strand

Can only move in the 5'-->3' direction

Cannot initiate synthesis from scratch, can only extend an existing nucleic acid chain (extends from the 3' end of the RNA primer)

Term
What is the sliding clamp and what does it do?
Definition
an accessory protein that helps hold the DNA polymerase onto the DNA strand for processive replication
Term
What is RNase H and what does it do?
Definition
A nuclease that recognizes RNA-DNA hybrid helix and removes the RNA that initially started the replication
Term
What is DNA ligase and what does it do?
Definition
links short stretches of DNA to form a continuous strand
Term
What is the lagging strand and why must there be one?
Definition

 the DNA strand that is synthesized discontinuously as a series of Okazaki fragments.

 

Because both template strands (anti-parallel) move through the replication factory in the same direction while the DNA polymerase can only synthesize in the 5' to the 3' direction

Term

What are telomeres?

 

Why does the chromosome need them?

Definition

the G-rich ends of chromosomes

 

Shortening occurs at the 5' end of DNA strands due to removal of RNA primers and polymerase not being able to fill in the gap without an existing chain to extend, if not for telomeres coding portions of the DNA could eventually be shortened out of the chain and lost.

Term
What does DNA Polymerase III do?
Definition
incorporates nucleotides to growing DNA chain
Term
What does DNA Polymerase I do?
Definition
replaces RNA primers with DNA
Term

What is telomerase and what does it do?

 

In what cells is it active?

Definition

an enzyme activity that repeatedly adds a sequence of six nucleotides to the 3' end of a DNA strand, using an enzyme-associated RNA molecule as a template; the catalytic subunit is a reverse transcriptase

 

active in rapidly proliferating cells, reactivates in cancer cells

Term

What do histones do?

 

Definition

interacts with DNA in a way that allows for very condensed storage of the DNA

 

Through covalent modification (acetylation, phosphorylation and methylation) they provide a mechanism for controlling access to the DNA and controlling gene expression

Term

Which portion of the histones are covalently modified?

 

Which residues are subject to which covalent modification?

Definition

the tails of the histones that are flexible, charged and extend out from the nucleosome

 

Lys- acetylation

Ser and His- phosphorylation

Lys and Arg- methylation

Term

Which form of chromatin is transcriptionally active?

 

Which is transcriptionally silent?

Definition

Euchromatin

 

Heterochromatin

Term
What makes up a nucleosome?
Definition

the eight histone proteins (two of each) H2A, H2B, H3 and H4

Approx. 146 base pairs wrap around the octet

the histone H1 binds outside the core

Term
How is DNA covalently modified?
Definition
Methylation of cytosine groups can potentially interfere with interaction of DNA-binding proteins which means it may be a mechanism for marking or "silencing" DNA that contains no genes; variations in methylation may be responsible for imprinting- the level of expression of gene depends on parental origin
Term
What molecule transfers the methyl group to the cytosine in eukaryotes?
Definition
S-adenosyl methinonine (SAM)
Term
What are the different types of DNA repair?
Definition

Mismatch repair

 

Base excision repair

 

Nucleotide excision repair

 

Double-strand break repair

Term
How are mismatches repaired?
Definition
incorrect base pairs are detected by MutS in bacteria (a homolog of MutS is found in humans) that binds to and bends the DNA at the mismatch, allowing an endonuclease to cleave the strand; a third protein unwinds helix so mispairing can be destroyed and polymerase can correctly fill in the gap
Term
How does base excision repair occur?
Definition

damaged base is removed by glycosylases leaving an abasic site. An endonuclease then cleaves the remaining backbone so that polymerase may fill in the gap

 

Glycosylases bind the DNA and cause the damaged base to flip out from the helix so that it can bind in the cavity on the protein surface, various side chains will take the place of the base until the endonuclease binds

The endonuclease (APE1 in humans) nicks backbone at 5' end of base and inserts two protein loops into the DNA to bend and expose abasic site, during the hydrolysis, an Mg2+ helps stablize.

The endonuclease stays with DNA until polymerase fills in the gap then ligase links the backbones

Term
How does nucleotide excision repair occur?
Definition
a damaged base is detected then it and approx. 30 of its neighbors are removed and the resulting gap is filled in by Pol1 and gaps sealed by DNA ligase
Term

What is the defect that leads to Cockayne syndrome?

What are the symptoms of Cockayne syndrome?

Definition

A defect in the proteins that recognize and remove stalled RNA polymerase (stalled due to DNA template damage). which means the DNA can't recognize the damage and definitely can't repair it, leading to apoptosis(cell death)

 

Symptoms: neural underdevelopment, failure to grow, and sensitivity to sunlight

Term

What defect leads to Xeroderma Pigmentosum?

 

What characterizes it?

Definition

Caused by mutations in genes directly involved in nuclease excision activity; proteins can detect damaged DNA but lack ability to repair it, means the cell does not undergo apoptosis, but damaged DNA can't be repaired

 

Characterized by sensitivity to sunlight (1000x more likely to get skin cancer than CockSyn) but do not have developmental problems

Term

Why does apoptosis occur?

 

Definition
The cell has detected damage it cannot correct; if the cell cannot detect and address the issues or properly start apoptosis uncontrolled proliferation and/or cancer may occur
Term
What are oncogenes?
Definition
go in and ask her
Term
What is p53 and what's so special about it?
Definition

tumor suppressor gene; transcription factors targets genes for cell cycle arrest, apoptosis, senescence, DNA repair or metabolic changes

 

approx. 50% of human cancer due to mutations of p53, rest of cancer due to mutations in p53 pathway: increase inhibitors, reduce activators or inactivation of downstream targets

Term
What do defects in p63 lead to?
Definition

defect in DNA binding domain leads to developmental disorders, cleft lip/palete; split hand/foot malformation, etc

 

defect in protein interaction domain leads to other developmental disorders

 

has a role in DNA damage induced apoptosis

Term
What is BRAC1 and what do defects lead to?
Definition

gene that dimerizes with BARD1 and has E3 ligase activity, works in pathway for DNA repair and homologous recombination;

 

defects lead to breast cancer, ovarian, pancreatic and prostate cancers, accounts for 30-60% of hereditary cancer

Term
What are Fanconi Anemia proteins?
Definition
same family as BRCA1 and RAD51, function in DNA repair, homologous recombination
Term
What is the funtion of retinoblastoma protein?
Definition

regulates E2F (transcription factor)- expression of cyclins and progression from S to G1 phase

 

loss of Rb leads to uncontrolled expression

Term
What are the 10 hallmark features of a cancer cell?
Definition

1. Self-sufficiency- cancer cells behave like arrogant, self-important egomaniacs. only listen to internal voice, and despite their anti-social behavior, they continue to carry on

 

2. Insensitivity: ignore signals from normal cells

 

3. Invasiveness: movement of cancer cells- metastasis is actually what makes caner a deadly disease

 

4. Immortality: every cell division picks up a few errors in DNA, cancer cells ignore all signals of damaged DNA and continue to divide

 

5. Greed: grow new blood vessels (angiogenesis) to get nutrients to grow

 

6. Cheating Death: ignore signals for apoptosis

 

7. Deregulated Energy Supplies: have different energy metabolism than normal cells

 

8. Masters of Disguise: escape detection by the immune system

 

9. Instability of DNA (under debate of hallmarked-ness): DNA of cancer cells constantly mutating and evolving, scrambling DNA into interesting state

 

10. Inflammation: not hallmark feature, but an effect on surrounding tissue

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