DNA, DNA Packing, and Nucleus

some types of bactriophages carry single-stranded DNA as their genetic material…

and many carry genetic material as RNA

Retroviruses (RNA viruses) are important to human health, why?

conveert their RNA genomes to DNA by reverse transcription

Erwin Chargaff showed that…

the DNA from different cells of a given species has the same percentage of each of the four baes

base composition varies…

among species

What are Chargaff’s rules?

for all DNA samples, the amount of A = T and the amount of G = C

Structure of DNA

• antiparallel

• double-stranded polymer of deoxyribonucleotides

• double helix

A specific purine (A or G) lines up with

its complementary pyrimidine (T or C) on the other strand

The sequence of one strand of DNA can be re-created by using the other strand as a…

template for replication

The strands of DNA are twisted around each other so that there is….

a major groove and a minor groove

phosphodiester bonds that join the 5’ carbon of the one nucleotide to the 3’ carbon of the next are oriented…

in opposite directions in the two DNA strands; ie antiparallel

How is DNA length measured?

in base pairs (bp)

DNA length is genergally referred to in…

kilobases (kb)

what are kilobases

multiples of 1000 base pairs

B-DNA

• majority of cellular DNA

• right-handed helix

• clear major and minor grooves

• 0.34nm per base pair

Z-DNA

• Transiently observed localized to specific DNA regions

• left-handed helix

• disordered

• significance unclear

A-DNA

• favored helix of dsRNA

• not a significant form of DNA

• right-handed helix

• wider minor groove

• narrower major groove

positive supercoil

twisted DNA that is twisted even further in the same direction

negative supercoil

twisting the DNA in the opposite direction that it is already coiled

Where does supercoiling occur?

In both linear and circular DNA molecules

• easily studied in circular DNA

A DNA molecule can go back and forth between…

the supercoiled state and the nonsupercoiled/relaxed state

What does extensive supercoiling do?

helps make chromosomal DNA more compact

What is topoisomerase role with supercoils?

can both induce and relax supercoils

Type I topoisomerases

introduce transient single-strand breaks in DNA

Type II topoisomerases

introduce double-strand breaks

example of type II topoisomerase

in bacteria, DNA gyrase

What binds DNA strands together?

relatively weak noncovalent bonds/interactions

DNA denaturation or melting can be induced….

experimentally by raising temperature or pH

How can process of DNA denaturation be monitored?

through light absorption since single and double-stranded DNA differ in their light absorbtions

All DNA absorbs light, with a maximum…

around 260nm

What happens to absorbance as DNA strands separate?

it increases rapidly

DNA melting temperature or Tm

the temperature at which half of the absorbance change is reached

what does the Tm vary on?

depends on A-T content

DNA renaturation (reannealing)

reformation of the DNA double helix

How is renaturation accomplished?

By lowering the temperature to permit hydrogen bonds to reform

nucleic acid hybridization

nucleic acids can be identified based on sequence

How does nucleic acid hybridization work?

Denatured DNA is incubated with a purified single-stranded DNA (a probe) with a sequence complementary to the sequence one is trying to detect

When bound to proteins, DNA….

is converted into chromatin

nucleoid

the DNA molecule is bound to small amounts of protein and localized to a region of the bacterial cell, this is that region

bacterial DNA is negatively…

supercoiled and folded into loops

plasmids

small, usually circular DNA molecules containing genes for their own replication

most plasmids are…

• supercoild

• replicate autonomously

• replication is somewhat synchronous with the chromosome

Histones

group of small basic proteins with high lysine and arginine content

• negatively charged DNA binds stably to positively charged proteins

five main types of histones

• H1

• H2A

• H2B

• H3

• H4

Chromatin contains about equal numbers of all of the types of histones except…

HI, which is present in about half the amount of the others

Histone octamers contained two…

H2A-H2B dimers and two H3-H4 dimers, with the DNA wrapped around the octamer

core particle

the octomer with 146 pb of DNA

linker DNA

extra DNA from the original 200 bp

first step of packaging of nuclear DNA

nucleosome formation

Isolated chromatin measures about

10 nm in diameter

chromatin of intact cells measures

about 30 nm

What does Histone H1 do?

facilitates formation of the 30-nm fiber

The 30-nm fiber seems to be packed together in…

an irregular, three-dimensional zigzag structure

The loops are spatially arranged through …

attachment to nonhistone proteins that form a chromosomal scaffold

The 30-nm fibers fold into DNA loops which are

50,000 - 100,000 bp in length, stabilized by cohesion protein

nucleosomes are packed together to form…

chromatin fibers and chromosomes

transcriptionally active DNA is less…

tightly packed than inactive DNA

Why does transcriptionally active DNA being looser make sense?

because the packaging would allow easy access by proteins involved in gene transcription

DNA packing ratio

the extent to which DNA has been folded

what do altering histones do?

they tightly regulate the portions of chromatin that are active or inactive

Each histone has a protruding tail that can be…

tagged by the addition of methyl, acetyl, phosphate, or other groups

histone code

the various combination of tags on the histone tail

histone methyltransferase

an enzyme that catalyzes the tagging reaction for methylation of lysine

what does methylation serve as a signal for?

activation or repression of transcription depending on the lysine involved

histone acetyltransferases (HATs)

enzyme that accomplishes the actylation of histone side chains

histone deacetylase (HDAC)

enzyme that accomplishes the opposite function of acetylation of histone side chains

an open chromatin is

active

a closed chromatin is

inactive

proteins with chromodomains are associated with

methylated histones charactristic of compact chromatin

proteins with bromodomains bind to

acetylated DNA associated with “open” chromatin

chromatin remodeling proteins

alter the position of nucleosomes along DNA

SWI/SNF family

an important class of remodelers, they slide nucleosomes or remove then from a region of chromatin, making the DNA more accessible

heterochromatin

sections of chromatin so highly compacted that they show up as dark spots in micrographs

euchromatin

loosely packed, diffuse chromatin

much of chromatin in metabolically active cells is euchromatic, but in preparation for cell division…

all the chromatin becomes highly compacted

after replication, each chromosome is composed of…

two identical chromatids

faculative heterochromatin can be…

converted to euchromatin and vice versa

constitutive heterochromatin

heterochromatin that is permanently compacted, it serves structural functions within chromosomes

two important types of heterochromatin

• centromeres

• telomeres

centromeres

• they maintain sister chromatid cohesion

• have attachment sites (kinetochores) for microtubules durig mitosis and meiosis

telemeres

• at tips of chromosomes

• two per chromosome

• protect ends of linear chromosomes from shortening during replication

• same sequence in every vertebrate studied

Eukaryotes have variable amounts of repeated DNA….

in their genomes, the rest is nonrepeated DNA

two categories of repeated DNA

• tandemly repeated DNA

• interspersed repeated DNA

most interspersed repeated DNA consists of familes of…

transposable elements (transposons) which can move around the genome and leave copies of themselves behind

the most abundant transposable elements are called…

LINEs (long interspersed nuclear elements)

LINEs are…

• 6000-8000 bp long

• contain genes required for their own mobilization

SINEs are…

• short interspersed nuclear elements

• less than 500 bp

• rely on enzymes from other elements for their movement

What are the most common SINEs in humans?

Alu sequences, they account for 10% of the human genome

Mitochondria and chloroplasts have their own…

chromosomes, which are devoid of histones and are usually circular

Both mitochondria and chloroplasts can encode some of their own…

polypeptides but depend on the nuclear genome to encode the rest of them

The nucleus is the site within the eukaryotic cell where the chromosomes are…

localized and replicated and the DNA they contain is transcribed

What is the nucleus bounded by?

a nuclear envelope

the nuclear envelop has what?

an outer and inner membrane separated by a perinuclear space

nuclear pores

specialized channels in the nuclear envelope where inner and outer membranes are fused

nuclear pores provide direct contact between…

the cytosol and the nucleoplasm (interior nuclear space)

What is the nuclear pore complex (NPC) built from?

about 30 different proteins called nucleoproteins

central granule

referred to as the transporter and is likely involved in moving molecules across the nuclear envelope

molecules enter and exit the nucleus through…

nuclear pores

small particles, less than 10 nm in diameter pass through pores at a rate…

proportional to the size of the particle

the NPC contains tiny…

aqueous diffusion channels through which small particles freely move