Mol Bio Lecture 3-Spring 2009-Post

8/8/2019 Mol Bio Lecture 3-Spring 2009-Post

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Molecular Biology Spring, 2009

Lecture 3: Higher Order DNA Structure

January 27th, 2009

Figure 4-20

Dr. Rich Dearborn

BRB 301B (x387) [email protected] Hours: Mon/Wed 1:30-2:30pm and by appointment

Dissociate

dna isspilling out

of nuc


2/23

Figure 4-21

Major Structural Features of Chromosomes and their Function

Telomere- specialized repeat sequence that helps maintainchromosome integrity and it serves to mark the life span of a

cell

Centromere-

specialized dnasequence that

cordinates separation

of replicated

chromosomes

(only 1 perchromosome)


3/23

Megagiantocorps new inhibitor of g1 cdk activitygenerizample 359 shows promis as anti tumorigenicagent. When treated with gz359 in early g1, which of thefollowing would be observed in the cell?

1) stimulate of origin firing

2) an inhibition of pre replication complex assemply

3) inhibitoon of dna synthesis

4) phosphorylation of Ifs

5) none of the above


4/23

Figure 4-72

- we have 2 meters of dna

needs to be packaged into 6

um nucleus

-Many proteins are involved inthe regulation of DNA higher

order structure we will focus

on 2 major classes of proteins

that medite this processinclude:

- topoisomerases- can

break and cut dna strands to

mediate compaction- histones- physical

scaffold around which dna is

compacted


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Berg et al. Fig 27.19

or

Supercoiling Generates Topoisomers

Lodish et al. Fig 4.7

Topoisomers are topologically identical

but geometrically distinct, differing

onlyin linking # (the # of complete turns

of the helix)

Negative supecoiling advantages

1. Condenses dna

2. Faciliates strand separation when

the supercoiling is relieved helpsreplication and transcription

3. Mediated by topoisomerases


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Figure 6-20aFigure 6-20b

Figure 6-20c

Both Compaction of DNA and Replication Processes Induce Supercoiling

With continued

movement a

topological knotwill form

Replication

machinery

for replicaiton

topoisomerases must

releive this supercoiling


7/23

Figure 5-21

During replication,

topoisomerases relieve

supercoiling to solve thewinding problem and prevent

toplogical knotting

If the dna ends are fixed how

can super coiling ve eliminted at

a local level ?? Must break the

dna molecule- sever one or both

strands in order to unwind dnaand allow for rotation


8/23

Fig 27.21


Topoisomerases alter the linking number to either

relax or supercoil dna by catalyzing a 3 step

process

a) cleave one or both strands of dna

b) pass a dna sement through the break

c) reseal the dna backbone

Type I topoisomerases cleave just one strand of

dna and function to relax ( a thermodynamically

favorable process)

Type II topoisomerases cleave both

strands of dna and us the energy of atp

hydolysis to add negative supercoild to

dna

Topoisomerases mediated this

interconversion (both directions)

type I relication

type II- compactionTyrosine is used in

nucleophilic attack to break

the phosphate backbone


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Topoisomerase I mechanism

Figure 5-22

-Critical tyrosine residue is found in the dnabinding cavity of topo I

-The hyroxyl group of the tyrosineparticipates in a nucleophilic attack on aphosphate group of the dna backbone to

form a phosphodiester linkage between the

enzyme and the dna which cleaves the dna

-The dna is now free to rotate around the

cleaved strand using the energy stored bc of

supercoiling

-The free hydroxyl group of the cleaved dna

strands then attacks the phosphotyrosineresidue to reseal the dna strand

-Topo I is a target for the anti cancer drug

camptothecin


10/23

Figure 5-23

Topoisomerase II mechanismT-segment

-T

he topo II dimer binds to DNA duplex segment (g segment)tightly and a second duplex segment (t segment) loosely

-Binding of atp to the 2 n terminal domains of topo II causes a

conformational change in topo II that 1) cleaves the g segment

and 2) facliliataes movement of the t-segment through the

central cavity

-Hydrolysis of atp re sets the enzyme with the g segment still

bound

-As for topo I, critical tyrosin residues mediate the process

-topoII is the target of the drugs novobiocin and cipro


11/23


Topoisomerase II Enzymes Mediate the Interconversion

Between Negatively Supercoiled and Relaxed DNA

-Many antibioticts that target topo IIcovalently link topo II to DNA

-This creates stalled replication forks that

trigger ATM induced apoptosis via p53 or

ds breaks that lead to the same result-Mutations in bacterial topo II enzymes

(ex DNA gyrase) lead to antibiotic

resistance

-In eukaryote topo II enzymes arecrititcalto replication and are the targets of

many anti-cancer drugs


12/23

Figure 4-1

Figure 4-9

The control of chromosome structure

and organization is critical to many cell

processes including:

-Packaging

-Replication

-Transcription


13/23

Figure 4-22

DNA is associated with proteins at all phases in the cell

cycle

The dna/protein complex is called chromatin

What are the protein components of chromatin? / structure/

fucntion of chromatin proteins?


14/23

Figure 4-23

The Basic Unit of Eukaryotic

Chromosome Structure is the

Nucleosome

Nucleosome contains:

-DNA

-Histones

-Topoisomerase II


15/23

Figure

4-31

Nucleosomes Mediate Access to

the DNA to Control Transcription

and Replication

Dna stuck in the middle there

is inaccessible

Other areas are exposed and

accessible

To read dna you must unwrap

it from a histone to access it


16/23

Figure 4-

33a

Nucleosome Structure

The nucleosome is composed of ~200

base pairs of dna and 2 coplies each of

histones h2a, h2b, h3 and h4 (the

histone octamer) bound to histone h1

The nucleosome core particle

consists of 147 bp of dna and the

histone octamer


17/23


Figure 4-27

Histones are positivley charged, allowing neg

charged dna to bind

-- wrapping of dna around histones reduces

its linear extent by 7 fold

-- the writhing of dna around the histone core

creates negative supercoiling (underwound

dna) when straightened out. The DNA can be

easily separated for transcription orreplication

--topo II a second major component of

chromatin is responsible for this winding


18/23

A critical feature of histones

are their amino terminal tails

which extend out from thecore structure

Histone amino-terminal tails are rich in

lysine and arginine residues that are

subject to covalent modification

Modification of histone tails plays a

critical role in modulating the affinity of

histones for DNA, which affects gene

expressionOne essential modification is the

acetylation of lysine residues by a class

of enzymes called histone

acetyltransferases (HATs)


19/23

Figure 4-33a

A critical feature of histones are their amino terminal tails

which extend out from the structure

-- histone amino-terminal tails are rich in lysine and arginineresidues that are subject to covalent modificatin

--modification of


20/23

Figure 4-44a

Figure 4-44b

The Histone Code

M = methylation

P- phosphorylation

A- acetylation

U- ubiqitination

The pattern (code) of

modificatuon

determines outcome


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Figure 4-45

Histone Modifications Control Access to DNA

Acetyllysine residues are

docking sites for proteins

containing bromodomains

such as transcription factos

that recruit rna polymerase

II the major enzyme oftranscription

Thus, histone aceylation

serves to activate genetranscription

In contrast, histone

deacetylces (HSACS)

contribute


22/23

Figure 4-43

Specific histone codes

direct chromatinremodeling complexes

that promote or silence

gene expression


23/23

Figure 4-57

Ultimatley, control of dna higher order structure

determines whether or not he information stored in dna

can be accessed

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