◦ DNA replication◦ DNA repair◦ DNA Organization

Key topics:

While functioning as a stable storage of genetic

information, the structure of DNA is far from static: ◦ A new copy of DNA is synthesized with high fidelity before

each cell division ◦ Errors that arise during or after DNA synthesis are

constantly checked for, and repairs are made◦ Segments of DNA are rearranged either within a

chromosome or between two DNA molecules giving offspring a novel DNA

DNA metabolism consists of a set of enzyme catalyzed and tightly regulated processes that achieve these tasks

The Meselson-Stahl experiment was about the origin of the two strands in each of the daughter genomes

Cells were grown on a medium containing only 15N isotope until all their DNA became fully 15N labeled

Cells were then switched to 14N medium and allowed to divide once

CsCl density gradient centrifugation was used to determine the mass of genomic DNA before and after each round of replication

The Meselson-Stahl experiment showed that the nitrogen used for the synthesis of new dsDNA becomes equally divided between the two daughter genomes

This suggests a semiconservative replication mechanism

Both strands are replicated simultaneously

Parental DNA strand serves as a template

Nucleotide triphosphates serve as substrates in strand synthesis

3’ HydroxylPrimer - the growing end of the chain makes a bond to the -phosphorus of nucleotide

Pyrophosphate is a good leaving group – separately hydrolysed to Pi irreversible

Polymerase I is most abundant but its primary function is in clean-up during replication, repair, and recombination

Polymerase II is probably responsible for DNA repair

Polymerase III is responsible for DNA replication

Initiation◦ Requires initiator proteins (trans-acting factors)

Elongation◦ Leading and Lagging strands (repeated priming)

Termination◦ Circular and linear chromosomes have unique

problems

DNA Primase Synthesizes Short RNA Primer Molecules on the Lagging Strand

Helicases - Open Up the DNA Double Helix in Front of the Replication Fork

Single strand binding proteins keep ssDNA out of trouble

Clamp subunits tether A Moving DNA Polymerase to the DNA

The Proteins at a Replication Fork Cooperate to Form a Replication Machine

Chemical reactions and some physical processes constantly damage genomic DNA◦ At the molecular level, damage usually involves changes in

the structure of one of the strands ◦ Vast majority are corrected by repair systems using the

other strand as a template◦ Some base changes escape repair and the incorrect base

serves as a template in replication◦ The daughter DNA carries a changed sequence in both

strands; the DNA has been mutated

Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most carcinogens are also mutagens

Mismatches arise from occasional incorporation of incorrect nucleotides

Abnormal bases arise from spontaneous deamination reactions or via chemical alkylation

Pyrimidine dimers form when DNA is exposed to UV light

Backbone lesions occur from exposure to ionizing radiation

The fundamental difference between

prokaryotes and eukaryotes is that

prokaryotes have a single type of

chromosome, while most eukaryotes

have a diploid number of chromosomes

of several different types in somatic cells

•The complete set of all metaphase chromosomes in a cell is called its karyotype

•Karyotypes are species specific, and cells of organisms within the same species will have the same karyotype

•Human karyotypes show chromosomes arranged in order according to size and position of the centromere

•Karyotypes allow geneticists to identify certain chromosome mutations that correlate with congenital abnormalities

Example of the human karyotypeExample of the human karyotype

11 22 33 44 55

66 77 88 99 1010 1111 1212

1313 1414 1515 1616 1717 1818

1919 2020 2121 2222 XX YY

• Certain regions called bands on chromosomes stain more intensely that other regions

• Banding patterns are specific for each chromosome and allow the chromosomes to be distinguished

• G-banding produces bands on chromosomes when they are stained with Giemsa stainChromosomes are first heat treated or subjected to proteolytic enzymes

• G bands reflect regions of DNA rich in AT residues (300 G bands distinguished in metaphase, 2000 G bands distinguished in prophase)

Example of the human karyotypeExample of the human karyotype

11 22 33 44 55

66 77 88 99 1010 1111 1212

1313 1414 1515 1616 1717 1818

1919 2020 2121 2222 XX YY

• Q-banding produces bands when chromosomes are stained with quinacrine dye, which binds preferentially to AT-rich regions of DNA

• In FISH (Fluorescence In Situ Hybridization), chromosomes are stained with fluorescent tags attached to specific DNA sequences

• Purpose of banding pattern: cytogenetic analysis and landmarks of locating genes (mapping genes)

Designations of Designations of the bands and the bands and interbands in the interbands in the human karyotypehuman karyotype

G Banding Pattern

http://www.bio.miami.edu/dana/250/karyocolor2.jpg

• An organism’s total DNA content is called its C-value: total amount of DNA in a haploid cell

• The amount of genetic material in a cell varies greatly among prokaryotes and eukaryotes

• A direct relationship does not exist between the C value and the structural or organizational complexity of the organism

• One reason for this is the variation in the amount of repetitive DNA sequences in the genome

No direct relationship No direct relationship between the C value and between the C value and the structural or the structural or organizational organizational complexity of the complexity of the organismorganism

• The large amount of DNA present in eukaryotic chromosomes is compacted by association with histones, forming structures called nucleosomes

• Nucleosomes fold further into chromatin fibers

• Each chromosome contains a large number of looped domains of 30-nm chromatin fibers attached to a protein scaffold

HISTONES

•Small basic proteins

•Constant amount in cells

•25% lysine & arginine (Net + charge)

•5 main types: H1, H2A, H2B, H3 & H4

•Equal amount of histones & DNA

•H2A, H2B, H3 & H4 are highly conserved among distinct species

•Histone proteins are among the most conserved proteins

•H1 varies in cells (in RBC it is replaced by H5)

NON-HISTONE PROTEINS

•All DNA chromosomal proteins minus histones

•Structural proteins or enzymes i.e. DNA replication enzymes, regulatory proteins, transcription factors…

•Differ in number and type in different cell types

•Acidic proteins (negatively charged)

•Equal amount of non-histones & DNA

•Example of HMGs (High-Motility Group proteins)

Bind to minor groove

Have a role in DNA bending

Have a role in formation of higher order chromatin structure

•Octamer of histones 2 (H2A, H2B, H3, H4) + linker histone H1 + 180 bp of DNA

•DNA compacts by winding 1 and ¾ turn of the outside of the histone octamer

•Under electron microscopy, 11 nm chromatin fiber (beads on a string)

Nucleosome StructureNucleosome Structure

Nucleosomes connected together by linker Nucleosomes connected together by linker DNA and H1 histone to produce the “beads-DNA and H1 histone to produce the “beads-on-a-string” extended form of chromatinon-a-string” extended form of chromatin

Packaging of nucleosomes into the 30-nm Packaging of nucleosomes into the 30-nm chromatin fiberchromatin fiber

The many different orders The many different orders of chromatin packing that of chromatin packing that give rise to the highly give rise to the highly condensed metaphase condensed metaphase chromosome (700 X chromosome (700 X compaction)compaction)

• The functional state of the chromosome is related to the extent of coiling

• The more condensed areas of the chromosome (heterochromatin) are genetically inactive

• The less compacted regions (euchromatin) contain genes that are expressed

• Constitutive vs facultative heterochromatin

• Centromeres are the sites at which chromosomes attach to the mitotic and meiotic spindles

• Consensus yeast centromeric region8bp-78 to 86 bp >90%AT-25bp

• The centromere region of each eukaryotic chromosome is responsible for accurate segregation of the replicated chromosome to the progeny cells during both mitosis and meiosis

The CentromereThe Centromere

• Telomeres are regions found at the end of chromosomes

• They are often associated with the nuclear envelope and are common to chromosomes of the same species

• Telomeres are needed for chromosome stability

Centromere and TelomeresCentromere and Telomeres