The document discusses several key topics related to DNA structure and function: DNA replication ensures each cell has an exact copy of the DNA before cell division. Errors are constantly checked and repaired to maintain high fidelity. DNA is also rearranged through processes like recombination. The tightly regulated enzymes that perform these metabolic processes were demonstrated by the Meselson-Stahl experiment to replicate DNA using a semiconservative mechanism. DNA is organized through various levels of compaction into condensed chromosomes. This dynamic structure, along with features like centromeres and telomeres, helps regulate DNA accessibility and proper segregation during cell division.

1. ◦ DNA replication
◦ DNA repair
◦ DNA Organization
Key topics:

2.  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

3.  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 15
N isotope until
all their DNA became fully 15
N
labeled
 Cells were then switched to 14
N
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

4.  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

5.  Both strands are replicated simultaneously

6.  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 α-
phosphorus of nucleotide
 Pyrophosphate is a good
leaving group – separately
hydrolysed to Pi irreversible

8.  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

9.  Initiation
◦ Requires initiator proteins (trans-acting factors)
 Elongation
◦ Leading and Lagging strands (repeated priming)
 Termination
◦ Circular and linear chromosomes have unique problems

10.  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

17.  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

18.  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

21. 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

22. •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

23. •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

24. 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

25. • 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 stain
Chromosomes are first heat treated or
subjected to proteolytic enzymes
• G bands reflect regions of DNA rich in AT
residues (300 G bands distinguished in

26. 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

27. • 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)

28. Designations ofDesignations of
the bands andthe bands and
interbands in theinterbands in the
human karyotypehuman karyotype
G Banding Pattern

31. • 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

32. No direct relationshipNo direct relationship
between the C value andbetween the C value and
the structural orthe structural or
organizationalorganizational
complexity of thecomplexity of the
organismorganism

33. • 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

34. HISTONES
•Small basic proteins
•Constant amount in cells
•25% lysine & arginine (Net + charge)
•5 main types: H1, H2A, H2B, H3 & H4

35. •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)

36. 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

37. • 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

38. • 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)

39. Nucleosome StructureNucleosome Structure

40. Nucleosomes connected together by linkerNucleosomes 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

41. Packaging of nucleosomes into the 30-nmPackaging of nucleosomes into the 30-nm
chromatin fiberchromatin fiber

43. The many different ordersThe many different orders
of chromatin packing thatof chromatin packing that
give rise to the highlygive rise to the highly
condensed metaphasecondensed metaphase
chromosome (700 Xchromosome (700 X
compaction)compaction)

45. • 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

46. • Centromeres are the sites at which
chromosomes attach to the mitotic and
meiotic spindles
• Consensus yeast centromeric region
8bp-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

47. The CentromereThe Centromere

48. • 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

49. Centromere and TelomeresCentromere and Telomeres