Molecular Biology – AP Biology

the blueprint of life [11]: DNA replication 1

DNA replication

Definition: the process of copying a parental DNA molecule to form two daughter DNA molecules.

Introduction to DNA replication

DNA replication is essential for cell proliferation, i.e. mitosis, meiosis.
DNA replication is a complex endeavor involving a series of enzyme activities.→see “DNA polymerases”
DNA replication is performed in a semiconservative and semidiscontinuous mode.→see “DNA replication is semi-conservative”
DNA replication has 3 stages: initiation, elongation and termination.→next section
DNA replication is tightly regulated, involving various protein-protein, protein-DNA interactions.
DNA replication of prokaryote and eukaryote shares similar features, but is distinctive in details.

Chemical Reaction of DNA replication

Essentials

1. Substrate: deoxynucleoside triphosphates(dNTPs)
2. Template: a primer-template junction

DNA is synthesized by extending the 3’ end of the primer (free 3’-OH is required)

– RNA primer or priming from a nick in DNA

3. Enzymes: DNA polymerases etc

4. Energy supply: Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis

5. Ions involved: Mg++ or Zn++

DNA polymerases

DNA polymerase I
- Pol I was the first enzyme discovered with polymerase activity, and it is also the best characterized one.
- Although abundant in cells (400/cell), Pol I is NOT the primary enzyme involved with bacterial DNA replication.
- Main functions of Pol I: (1) Fill any gaps in the new DNA that result from the removal of the RNA primer by its 5’ -3’ polymerase activity; (2) Remove a new mispaired base by proofreading (校读)3’-5’ exonuclease (外切酶) activity. (3)Remove the RNA primer by its 5’-3’ exonuclease activity

The 3′–>5′ exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. (source)

DNA polymerase III
- The primary polymerase in DNA replication, although lower in abundance (15/cell)than pol →referred to as “replicase”
- functions: (1) 5’→3’ polymerase activity; (2) 3’→5’ exonuclease activity – proofreading
- Catalytic efficiency: much higher than pol I→High processivity and polymerization rate
- A multi-unit complex: “holoenzyme” (全酶)

DNA replication is semi-conservative

Bet you all have learned it in high school, and the famous experiment by Meselson and Stahl. We still need to go over the points again as they are essentially important for what we will learn next.

The key to the mechanism of DNA replication is the fact that each strand of the DNA double helix carries the same information-their base sequences are complementary (we talked about this in THE BLUEPRINT OF LIFE [2]: PRIMARY AND SECONDARY STRUCTURE OF DNA).

During replication, the two parental strands separate and each acts as a template (that’s right, the template for DNA replication is DNA itself!)to direct the enzyme-catalyzed synthesis of a new complementary daughter strand with the normal base-pairing rules (A-T, C-G)

This semi-conservative mechanism was demonstrated experimentally in 1958 by Meselson and Stahl.

Hypotheses:

In the experiment:

E. coli cells were grown for several generations in presence of the stable heavy isotope ¹⁵N so that their DNA became fully density labeled (both strands are ¹⁵N labeled: ¹⁵N/¹⁵N)

The cells were then transferred to medium containing only normal ¹⁴N and, after each cell division, DNA was prepared from a sample of the cells and analyzed on a CsCI gradient using the technique of equilibrium (isopycnic) density gradient centrifugation, which separates molecules according to differences in buoyant density.

After the first cell division, when the DNA had replicated once, it was all of hybrid density, in a position on the gradient half way between fully labled (¹⁵N /¹⁵N )and fully light (¹⁴N/¹⁴N). After the second generation in ¹⁴N, half of the DNA was hybrid density and half fully light.

Thus, two of the hypotheses were denied, left us with the semi-conservative mechanism.

After each subsequent generation, the proportion of ¹⁴N/¹⁴N increased, while some DNA of hybrid density persisted. Thus the semi-conservative mode of DNA replication is confirmed: each daughter molecule contains one parental strand and one newly-synthesized strand.

DNA replication & PCR, General Biology, Open Courses at UC-Berkeley

General Biology, Great open courses given by Gary L. Firestone, Michael Meighan Jasper D. Rine and Jennifer A. Doudna, professors at UC-Berkeley.

There is more to DNA replication than we talked about yesterday so I tried to upload this video to help you learn more.

“tried? ”

“Well, turned out ‘ DNA replication and PCR open course at Berkeley.mp4 exceeds the maximum upload size (8 MB)for this site.'”

So I would just provide the link where you can watch it online.

After watching it, maybe you will find more than just DNA replication: you may as well find how it feels going to college.

I look forward to it every time I have finished an open course online. Hope you do, too.

>>click here to watch the video

the blueprint of life [10]: eukaryotic structure of DNA 3

let’s go over the terms again. Make sure you all know what they mean.

•Nucleus: 细胞核; Nucleolus: 核仁; Nucleoid: 类核

• Mitosis: 有丝分裂; Meiosis: 减数分裂

Interphase: 分裂间期; Prophase: 分裂前期; Metaphase: 分裂中期; Anaphase: 分裂后期; Telophase: 分裂末期

• Histone: 组蛋白

• Nucleosome: 核小体

•Chromosome: 染色体; Chromatin: 染色质; eu- 真染色质; hetero- 异染色质

Sister chromotid 姐妹染色单体;
mitotic spindle 纺锤体
spindle microtubule纺锤丝

• Centromere: 中心粒; Telomere: 端粒

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The familiar picture of a chromosome is actually that of the most highly condensed state at mitosis(which we reviewed in the blueprint of life [8]: eukaryotic structure of DNA 1(chromatin structure)).

As the daughter chromosomes are pulled apart by the mitotic spindle at cell division, the fragile centimeters-long chromosomal DNA would certainly be sheared by the forces generated, were it not in this highly compact state.

picture above is from Instant Notes in Molecular Biology

As we can see from the picture, the chromosomal loops fan out from a central scaffold or nuclear matrix region consisting of protein(which we talked about last section). One possibility is that consecutive loops may trace a helical path along the length of the chromosome.
The centromere is the constricted region where the two sister chromatids are joined in the metaphase chromosome. This is the site of assembly of the kinetochore, a protein complex which attaches to the microtubules of the mitotic spindle.

(The microtubules act to separate the chromotids at anaphase)

The DNA of the centromere has been shown in yeast to consist merely of a short AT-rich sequence of 88 bp, flanked by two very short conserved regions, although in mammalian cells, centromeres seem to consist of rather longer sequences, and are flanked by a large quantity of repeated DNA, known as satellite DNA.

Telomeres are specialized DNA sequence that form the ends of the linear DNA molecules of the eukaryotic chromosomes. A telomoere consists of up to hundreds of copies of a short repeated sequence(5′-TTAGGG-3′ in humans), which is synthesized by the enzyme telomerase in a mechanism independent of normal DNA replication.

The telomeric DNA forms a special secondary structure, the function of which is to protect the ends of the chromosome proper from degradation.(Independent synthesis of the telomere acts to counteract the gradual shortening of the chromosome resulting from the inability of normal replication to copy the very end of a linear DNA molecule—we will talk about this when reaching DNA replication)

Interphase chromosome

In interphase(S phase, to be exact), the genes on the chromosomes are being transcribed and DNA replication takes place. During this time, the chromosmes adopt a much more diffuse structure and cannot be visualized individually. It is believed, however, that the chromosomal loops are still present, attached to the nuclear matrix.

The blueprint of life [9]: eukaryotic structure of DNA 2(nucleosome)

Eukaryotic chromosome is packaged in hierarchical levels, mediated by various proteins.

DNA duplex→Nucleosome → Chromatin →Chromosome

We talked about DNA duplex and chromatin; now it’s time to meet the basic unit of chromatin structure–nucleosome.

Definition: nucleosome is the chromatin subunit that consists of DNAand a set of eight histone core proteins(complex of (H2A)₂(H2B)₂(H3)₂(H4)₂, →octamer. More loosely with one molecule of H1 )

Comparison

The proteins protect the DNA from the action of micrococcal nuclease.

Micrococcal Nuclease is an endo–exonuclease that preferentially digests single-stranded nucleic acids The enzyme is also active against double-stranded DNA and RNA and all sequences will be ultimately cleaved.(wikipedia)

Digestion with nuclease leads to the loss of H1, yielding a very resistant structure consisting of 146 bp of DNA associated very tightly with the histone octamer. The structure, known as the nucleosome core, is structurally very similar whatever the source of the chromatin.

Adjacent nucleosome is connected by a varied length (10-100 bp, average 55bp)of DNA, called “linker DNA”

One molecule of linker histone H1 binds to the linker DNA between nucleosome.

H1 also acts to stabilize the point at which the DNA enters and leaves the nulceosome core.

↑Chromatins at different packaging levels

30 nm chromatin fiber: condensed form

10 nm chromatin fiber : less-condensed form, like a thread of beads

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Summary of Nucleosome Structure:

NUCLEOSOME STRUCTURE, image from bricker.tcnj.edu

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Higher Order Structure

The organization of chromatin at the highest level seems rather similar to that of prokaryotic DNA(see THE BLUEPRINT OF LIFE [7]: PROKARYOTIC CHROMOSOME STRUCTURE OF DNA). Even the size of the loops is approximately the same, up to aorund 100 kb of DNA, although there are many more loops in a eukaryotic chromosome.

The loops are constrained by interaction with a protein complex known as the nuclear matrix. The DNA in the loops is in the form of 30 nm fiber, and the loops form an array about 300 nm across.

”Solenoid model “of 30-nm chromatin fiber

6 nucleosomes per turn

“Zigzag model” of 30-nm chromatin fiber

6 nucleosomes per turn, longer linker DNA may be required

HIGHER ORDER STRUCTURE, shown by prof. Dong

the blueprint of life [8]: eukaryotic structure of DNA 1(chromatin)

• Mitosis: 有丝分裂; Meiosis: 减数分裂

Interphase: 分裂间期; Prophase: 分裂前期; Metaphase: 分裂中期; Anaphase: 分裂后期; Telophase: 分裂末期

• Histone: 组蛋白 hhistidine 组氨酸

• Nucleosome: 核小体

•Chromosome: 染色体; Chromatin: 染色质; eu- 真染色质; hetero- 异染色质

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The total length of DNA in a eukaryotic cell depends on the species, but it can be thousands of times as much as in a prokaryotic genome.

Eukaryotic chromosome is made up of a number of discrete bodies called chromosomes. The DNA in each chromosome is believed to be a single linear molecule, which can be up to several centimeters long.

All these each contain a long linear DNA molecules, which must be packaged into the nucleus, a space of approximately the same volume as a bacterial cell

SO, much longer DNA chains packaged into a space of the same volume as a bacterial cell? → for example, 2 cm of DNA length versus ~10 µm of cell size for fruit fly; most condensed form of human chromosome is about ~2 µm long = 10,000× packing ratio

the obvious result is in their most highly condensed forms, the chromosomes have an enormously high DNA concentration: perhaps 200 mg/ml.!

The feat of packing is accomplished by the formation of a highly organized complex of DNA and protein, known as the chromatin, a nucleoprotein complex. (←our hero today, has finally showed up.)

Chromosomes greatly alter their level of compectness as cells progress through the cell cycle, vary between highly condensed chromosomes at metaphase(just before the cell division), and very much more diffuse structures in interphase.(This implies the existence of different levels of organization of chromatin)

More than 50% of the mass of chromatin is protein. Most of the protein in eukaryotic chromatin consists of histones, of which there are five families: H2A, H2B, H3 and H4, known as the core histones, and H1.

The core histones are small proteins, with masses between 10 and 20 kDa, and H1 histones are a little larger at around 23 kDa.

The unified atomic mass unit (symbol: u) or dalton (symbol: Da) is the standard unit that is used for indicating mass on an atomic or molecular scale (atomic mass). One dalton is approximately the mass of a nucleon and is equivalent to 1 g/mol.^[1] It is defined as one twelfth of the mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state,^[2] and has a value of 1.660538921(73)×10⁻²⁷ kg.^[3]

All histones proteins a large positive charge; between 20 and 30% of their sequences consist of the basic amino acids, lysine and arginine. This means that histones will bind very strongly to the negatively charged DNA in forming chromation.

amino acids in English?

Members of the same histone class(family) are very highly conserved between relatively unrelated species, for example between plants and animals, which testifies to their crucial role in the chromation.

Within a given species, there are normally a number of closely similar variants of a particular class, which may be expressed in different tissues, and at different stages in development.

There is not much similarity in sequence between the different histone classes, but structural studies have shown that the classes so share a similar tertiary structure, suggesting that all hisotnes are ultimately evolutionarily related.

H1 histones are somewhat distinct from the other histone classes in a number of ways; in addition to their larger size, there is more variation in H1 sequences both between and within species than in other classes. Histone H1 is more easily extracted from bulk chromatin, and seems to be present in roughly half the quantity of the other classes, of which there are very similar amounts.

Next section we will cover the distinct role of histone Hi in chromatin structure.

the blueprint of life [7]: prokaryotic chromosome structure of DNA

First, let’s make sure the anatomy of prokaryotes are familiar to us:

Prokaryotes are the simplest living cells, typically 1~10μm in diameter, and are found in all environmental niches from the guts of the animals to acidic hot springs.

They are bounded by a cell (plasma)membrane comprising a lipid bilayer, in which are embeded proteins that allow the exit and entry of small molecules.
Most prokaryotes also have a rigid cell wall outside the plasma membrane, which prevents the cell from swelling or shrinking in environments where osmolarity differs significantly from that inside the cell.
Sometimes the cell wall is surrounded by an (often) polysaccharide envelope called capsule.
The cell interior (cytoplasm or cytosol) usually contains a single, circular chromosome compacted into a nucleoid and attached to the membrane.
There are no distinct subcelluar organelles in prokaryotes as in eukaryotes(except for the ribosomes核糖体).
The surface of a prokaryote may carry pili, which allow the prokaryote to attach to other cells and surfaces. Some prokaryotes also carry flagella, whose rotating motion allows the cell to swim.

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To talk about prokaryotic chromosome structure, we use E. coli (大肠杆菌)as the example.

E. coli chromosome contains a single supercoiled circular DNA molecule of length 4.6 million bp.
E. coli chromosome is highly folded: 1500 µm of DNA length versus ~1 µm of cell size, forming a structure called the nucleoid.
The nucleoid has a very high DNA concentration, perhaps 30~50 mg/ml, as well as containing all the proteins associated with DNA, such as polyerases, repressors(a protein that is determined by a regulatory gene, binds to a genetic operator, and inhibits the initiation of transcription of messenger RNA), etc.

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DNA domains (loops)

Remember this famous electron micrograph of an E. coli cell we showed before? The cell was carefully lysed, all the proteins removed and then, it was spread on an EM grid to reveal all of its DNA.

Several of such experiments revealed one level of organization of the nucleoid.

The DNA consists of 50~100 domains or loops, about 50~100 kb in size (kb: kilobase, a unit of measure of the length of a nucleic-acid chain that equals one thousand base pairs).
The ends of the loops are constrained by binding to a structure which probably consists of proteins attached to part of the cell membrane.
Not known whether the loops are static or dynamic, but one model suggests that the DNA may spool(wind) through sites of polyerase or other enzymic action at the base of the loops.

E coli DNA instant notes

image above is from”Instant Notes in Molecular Biology”

Supercoiling of the genome

The E. coli chromosome as a whole is negatively supercoiled, although there is some evidence that indicates individual domains may be supercoiled independently. Electron micrographs indicate that some domains may not be supercoiled, perhaps because the DNA has become broken in one strand, where other domains clearly do contain supercoils.

The domains may be topologically independent. There is, however, no real biochemical evidence for major differences in the level of supercoiling in different regions of the chromosome in vivo

DNA-binding proteins

The looped DNA domains of the chromosome are constrained further by inter-action with a number of DNA-binding proteins.

The most abundant of these are protein HU, a small basic (+charged) dimeric protein, which binds DNA non-specifically by the wrapping of the DNA aorund the protein.

And H-NS (formerly known as the protein H1), a monomeric neutral protein, which also binds DNA non-specifically in terms of sequence, but seems to have a preference for regions of DNA which are intrinsically bent.

These proteins are sometimes known as histone-like proteins, and have the effect of compacting the DNA, which is essential for the packaging of the DNA into the nucleoid, and of stabilizing and constraining the supercoiling of the chromosome.

the blueprint of life [6]: Chromosomal Structure of DNA 1

vocabulary

•Nucleus: 细胞核; Nucleolus: 核仁; Nucleoid: 类核

• Mitosis: 有丝分裂; Meiosis: 减数分裂

Interphase: 分裂间期; Prophase: 分裂前期; Metaphase: 分裂中期; Anaphase: 分裂后期; Telophase: 分裂末期

• Histone: 组蛋白

• Nucleosome: 核小体

•Chromosome: 染色体; Chromatin: 染色质; eu- 真染色质; hetero- 异染色质

• Centromere: 中心粒; Telomere: 端粒

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WHAT is chromosome:

Structure containing the genes of a cell and made of a single DNA molecule and its associated proteins.

CHROMOSOMES OF EUKARYOTES, shown by prof. Dong

HOW long is DNA in an chromosome
HOW LONG IS DNA, shown by prof. Dong.

→A chromosome is too long to fit into a cell without compaction.

WHY is DNA packed into chromosomes

Chromosome is a compact form of the DNA that readily fits inside the cell
To protect DNA from damage
DNA in a chromosome can be transmitted efficiently to both daughter cells during cell division
Chromosome confers an overall organization to each molecule of DNA, which regulates gene expression as well as recombination

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Diversity of chromosomes

in terms of:

Shape: circular or linear
Number: species-specific

eg. -fruitfly 8; -human 46; -horse 64; -dog 78; -chicken 78

-maize 20; -rice 24; -wheat 42

Copy number: haploid单倍体, diploid双倍体, polyploid 多倍体

Overall structure: highly different between prokaryotes 原核生物and eukaryotes真核生物

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Next section we will first talk about prokaryotic chromosome structure of DNA.

The blueprint of life [5]: spectroscopic and thermal properties of DNA

UV absorption

DNA absorbs UV light due to the conjugated aromatic nature of the bases; the sugar-phosphate backbone does not contribute appreciably(perceptibly/measurably) to absorption.

The wavelength of maximum absorption of light by both DNA and RNA is 260 nm, which is conveniently distinct from the λmax of protein(280 nm).

The absorption properties of DNA can be used for detection, quantitation and assessment of property.

Hypochromicity

UV absorption at 260nm is greatest for isolated nucleotides, intermediate for single-stranded DNA(ssDNA) or RNA, and least for double-stranded DNA(dsDNA)

The classical term for the change in absorbance is hypochromicity. For example, dsDNA is hypochromic (from the Greek for ‘less colored‘) relative to ss DNA, which is hypochromic relative to isolated nucleotides.

Thermodynamics of DNA

Denaturation: the transition of macromolecule from the native state to the denatured state. For DNA, under denaturing conditions (heating or high pH), double helix is separated to generate single-stranded form.

Melting Temperature(Tm): the temperature at which the rise in A260(absorbance at 260nm) is half complete during denaturation.

Factors that affect the Tm

1. G+C content: the higher G+C content, the higher Tm.

2. Ionic strength: The Tm increases as the cation (+) concentration increases. like Na+, K+ or Mg2+.

3. High pH or Agents that disrupt H-bonds or interfere with base stacking: formamide (甲酰胺)or urea (尿素)will decrease the Tm.

4. The imperfect hybridization between related but not completely complementary strands will reduce the Tm, about 1 °C for each percent mismatch.

The process of denaturation can be observed conveniently by the increase in absorbance as double-stranded nucleic acids are converted to single strands

Renaturation: Process of a macromolecule returning to its native 3-D structure. For DNA this involves the two strands of denatured DNA basepairing to restore the nature form of dsDNA. Also known as reannealing (重退火).

Requirements for renaturation:

1. Proper salt concentration: neutralize electrostatic repulsion

2. Proper reannealing temperature: 20-25℃ below Tm

Determinants for renaturation efficiency

(DNA renatures on cooling, but will form fully double-stranded native DNA only if the cooling is sufficiently slow to allow the complementary strands to anneal.)

1. Extent of base matching and

2. copy of matching regions

Thus, repetitive DNA renatures faster than single copy DNA

RENATURATION CURVES FOR E.COLI &MOUSE DNA, shown by prof. Dong

The blueprint of life [4]: Tertiary Structure of DNA

Finally, we come to the last part of the molecular structure of DNA.

4. Tertiary Structure of DNA – superhelix structure

Advanced folding and intertwining of DNA molecules over the secondary structure .

DNA topology

1. Linear DNA:

Commonly seen in eukaryotes,
with extreme length,
complementary sequence
included in the chromatin (the combination or complex of DNA and proteins that make up the contents of the nucleus of a cell)
interacting with other cellular components.

2. Circular DNA:

(DNA frequently occurs in nature as closed-circular molecules, where the two single strands are each circular and linked together. The number of links is known as the linking number(Lk).)

Usually seen in prokaryotes, e.g. plasmid (质粒), circular bacterial chromosomes and many viral DNA molecules
cccDNA (covalently closed circular DNA) → supercoiled or Relaxed; ncDNA (nicked circular DNA): a nick (缺刻)formed by breaking one phosphodiester bond
two complementary single strands are each joined into circles, 5′ to 3′, and are twisted around one another by the helical path of DNA.
The molecule has no free ends and the two single strands are linked together a number of times corresponding to the number of double-helical turns in the molecule.

cccDNA Topology

Supercoiled DNA

Negative superhelix: natural status of cccDNA with less intra- molecular tension (underwound effect)

Positive superhelix: unnatural status with higher tension (overwound effect)

Relaxed circular DNA: intermediate between negative superhelix and positive superhelix

Topological Equation of cccDNA

L = T + W

L=Linking number=total number of times one strand of the double helix links the other

T=Twisting number= the number of times one strand completely wraps around the other strand

W=Writhing number= the number of times that the long axis of the double helical DNA crosses over itself in 3-D space

Features

1. The linking number of a closed-circular DNA is a topological property, that is one which cannot be changed without breaking one or both of the DNA backbones. (A molecule of a given linking number is known as a topoisomer. Topoisomers differ only in their linking number)

2. Twisting number

For B DNA, T>0(10 bp per turn)

For A DNA, T>0(10.5 bp per turn)

For Z DNA, T<0 (12 bp per turn)

3. Writhing number

Relaxed: W=0

Negative supercoils: W<0

Positive supercoils: W >0

TWIST &WRITHE, from Instant Notes in Molecular Biology(3rd edition)

Topoisomerases (enzymes used to regulate the level of supercoiling of DNA molecules)

To alter the linking number of DNA, the enzymes must transiently break one or both stands. There are two classes of topoisomerases:

Type I Topoisomerase nick one strand of the DNA, changing 1 Linking-number at a time(+/-1 Lk)

Type II Topoisomerase, which requires the hydroloysis of ATP, break two strands of DNA, changing 2 Linking-number at a time(+/-2Lk); also able to unlink DNA molecules.

Most topoisomerases reduce the level of positive or negative supercoiling, that is, they operate in the energetically favorable direction. (However, DNA gyrase, a bacterial type II enzyme, uses the energy of ATP hydrolysis to introduce negative supercoiling into hence removing positive supercoiling generated during replication.)

Topoisomerases are essential enzymes in all organisms; they are involved in replication, recombination and transcription.

Both type I and II enzymes are the target of anti-tumor agents in humans.

DNA superhelix

Biological significance of Superhelix:

DNA packing:

Eg.

This is a famous electron micrograph of an E. coli cell that has been carefully lysed, then all the proteins were removed, and it was spread on an EM grid to reveal all of its DNA.

DNA functioning:

Negative supercoils serve as a store of free energy that aids in processes requiring strand separation, such as DNA replication and transcription; Strand separation can be accomplished more easily in negatively supercoiled DNA than in relaxed DNA.

1. DNA in cells is negatively supercoiled;

2.(-)supercoiling introduced by a topoisomerase II (gyrase 促旋酶) in prokaryotes and by nucleosome (核小体)in eukaryotes

3.Cruciform or bubble structures introduced by (-) supercoiling are potential protein-binding sites

>>click here to watch the video

Summary of Nucleosome Structure:

Higher Order Structure

vocabulary

Diversity of chromosomes

UV absorption

Thermodynamics of DNA

DNA topology

DNA superhelix

Thermodynamics of DNA