DNA and Chromatin Structure Lecture Notes PDF

BIOL-2120 INTRODUCTION TO CELL & MOLECULAR BIOLOGY Dr. Michael T. Klein ([email protected]) LECTURE 6 DNA & CHROMATIN STRUCTURE Reference text: Essential Cell Biology, 5th ed., Alberts et al. 2019. Chapter 5 Ima...

BIOL-2120 INTRODUCTION TO CELL & MOLECULAR BIOLOGY Dr. Michael T. Klein ([email protected]) LECTURE 6 DNA & CHROMATIN STRUCTURE Reference text: Essential Cell Biology, 5th ed., Alberts et al. 2019. Chapter 5 Images provided by M. T. Klein or W. W. Norton & Company unless stated otherwise MTK, 2025-01-30 (1)  CORRELATION OF DNA’S STRUCTURE AND FUNCTION  As the “master molecule” of life, DNA has to: Reliably carry the heritable information (the genotype) that underpin the basis of an organism’s phenotype (appearance, function, behavior, etc.) Be able to replicate itself so that cells can divide and the species can reproduce Have a mechanism for translating DNA sequences into amino acid sequences (a genetic code) Generate and preserve genetic variation by a mechanism that changes the information contained in genes (mutation) and a means to limit the effects of DNA damage (DNA repair) MTK, 2025-01-30 (2)  THE SEARCH FOR GENETIC MATERIAL  We’ll explore some of the history behind how DNA, its role in life, and its molecular structure were discovered. These investigations serve as a excellent examples of deductive reasoning and modeling of complex structures and processes based on rather limited observations from profoundly innovative experiments.  Scientists where originally unsure whether it was DNA or proteins that encoded genetic information  Consider the chemical and emergent properties of DNA vs. proteins: What properties of DNA made it ideal for storing and transmitting genetic information? What properties of proteins make it less than ideal for this role? What properties of these molecules could have made scientists think proteins were the genetic material rather than DNA? MTK, 2025-01-30 (3)  THE SEARCH FOR GENETIC MATERIAL DISCOVERY OF GENES  The concept of a gene was established by Gregor Mendel in the mid 19th century with his study of inheritance in pea plants; he gave us terms like genotype, phenotype, and: Gene – a heritable characteristic (e.g., flower color) Alleles – variations of a gene (e.g., different colors) Dominant trait – the trait that masks expression of a recessive allele (e.g., purple flowers) Recessive trait – the traits that is “masked” by the dominant allele (e.g. white flowers)  Inheritance and gene expression are far more Genetics Introductory Video: complicated than this, but the importance of https://drive.google.com/file/d/16Oy_MzpZE-gKrlM1Qnp2_9bLhmmWKwSq Mendel’s discoveries cannot be understated MTK, 2025-01-30 (4)  THE SEARCH FOR GENETIC MATERIAL THE GRIFFITH EXPERIMENT  In 1928, Frederick Griffith conducted an experiment that would give us our first clues to the molecular identity of the genetic material  He showed that heat-killed infectious bacteria can transform harmless live bacteria into pathogens Transformation is a term still used today that specifically refers to the uptake of exogenous DNA into bacterial cells The pathogenic strain (S-stain) could produce glycocalyx, giving it a capsule layer that protects it from the immune system The non-pathogenic strain (R-strain) lacked a capsule and would not harm the host An unknown component of the dead bacteria was able to transform other bacteria MTK, 2025-01-30 (5)  THE SEARCH FOR GENETIC MATERIAL FRACTIONATION EXPERIMENTS  To narrow down the possible type of molecules responsible for transformation, other researchers conducted fractionation experiments Cells are ruptured, homogenized, and different components are isolated ꟷ How might this be accomplished? This is still a common practice today when the causative agent of some phenomenon is unknown  Only the DNA fraction could transform the R-strain into an S-strain MTK, 2025-01-30 (6)  THE SEARCH FOR GENETIC MATERIAL HERSHEY & CHASE EXPERIMENTS  Viruses had been identified in the early 20th century in tobacco plants Something was infecting tobacco plants that was far smaller than any known bacteria This unknown pathogen was given the Latin name for poison: virus Viruses were known to be very simple  Hershey and Chase worked with bacteriophages – viruses that infect bacteria Bacteriophages are composed only of protein and DNA MTK, 2025-01-30 (7)  THE SEARCH FOR GENETIC MATERIAL HERSHEY & CHASE EXPERIMENTS  They labeled the viral protein and DNA with different radioisotopes and looked for the isotope(s) present in cells after they were infected by the virus  They found that only DNA entered the bacterial cells, this could mean only one thing: DNA was the genetic material, not protein How could they specifically label proteins and DNA with different radioisotopes if they didn’t know the structure of the virus yet? MTK, 2025-01-30 (8)  DETERMINING THE STRUCTURE OF DNA NUCLEOTIDES  After DNA was accepted as life’s genetic material, it could be isolated and studied chemically  DNA was known to be polymer of nucleotides, specifically deoxyribonucleotides  No matter what organism was studied, it was found that DNA was built out of only 4 different types of deoxyribonucleotides, each varying only in their nitrogenous base MTK, 2025-01-30 (9)  DETERMINING THE STRUCTURE OF DNA Purines Pyrimidines NITROGENOUS BASES  In 1950, Erwin Chargaff reported that DNA nitrogenous base composition varies from one species to the next  They did not have a means to sequence DNA yet, but from these simple chemical studies of DNA, Chargaff Adenine (A) Thymine (T) established two simple rules  Chargaff’s rules: Within an organism the amount of A = T and the amount of G = C, this never varies! The ratio of AT to GC varies between species but is largely consistent among members of a species  The basis for these rules was not understood until the discovery of the DNA double helix Guanine (G) Cytosine (C) MTK, 2025-01-30 (10)  DETERMINING THE STRUCTURE OF DNA FRANKLIN’S IMAGE REVEALED THE HELICAL STRUCTURE OF DNA  Maurice Wilkins and Rosalind Franklin used a technique called X-ray crystallography to study molecular structure  In 1952, Franklin produced an X-ray diffraction image of the DNA molecule (“Photo 51”)  Using Franklin's image as the crucial piece of evidence, Watson and Crick went on to publish their Nobel Prize-winning DNA structure Rosalind Franklin Video: https://drive.google.com/file/d/1FnSiNJvyCPF77lV5WBi37CJFALJaQwRW MTK, 2025-01-30 (11)  DETERMINING THE STRUCTURE OF DNA FRANKLIN’S IMAGE REVEALED THE HELICAL STRUCTURE OF DNA The width of the helix can be measured These diamonds showed the helix continued in a regular spiral with a consistent width The pattern of these The pitch of the helix can be smudges indicate a right- deduced and, with the width, handed helix the turning rate can be known Interactive Link: https://www.dnalc.org/view/15014-Franklin-s-X-ray-diffraction-explanation-of-X-ray-pattern-.html MTK, 2025-01-30 (12)  DETERMINING THE STRUCTURE OF DNA WATSON & CRICK PIECE TOGETHER THE STRUCTURE OF DNA  Franklin’s X-ray crystallographic images enabled Watson and Crick to deduce the geometry of DNA and, with the known chemical properties of nucleotides and DNA, they were able to assemble a structural model of DNA  The X-ray images let them deduce the width of the helix and the spacing of the nitrogenous bases  The pattern suggested that the DNA molecule was made up of two strands, forming a double helix MTK, 2025-01-30 (13)  DETERMINING THE STRUCTURE OF DNA WATSON & CRICK PIECE TOGETHER THE STRUCTURE OF DNA  At first, Watson and Crick thought the bases paired like with like – e.g., A with A, G with G, and so on  Such pairings did not result in a uniform width, and this did not match Franklin’s data showing DNA has a constant width  Instead, pairing a purine with a pyrimidine resulted in a uniform width, consistent with Franklin's data MTK, 2025-01-30 (14)  DETERMINING THE STRUCTURE OF DNA WATSON & CRICK PIECE TOGETHER THE STRUCTURE OF DNA Adenine (A) Thymine (T)  Based on Chargaff’s observation that in any organism the amount of A = T and the amount DNA of G = C, Watson and Crick deduced the Backbone correct base pairing: DNA Backbone A-T pairs have 2 hydrogen bonds Guanine (G) G-C pairs have 3 hydrogen bonds Cytosine (C)  How does this affect DNA separation at the start of DNA replication or transcription? DNA Backbone DNA Backbone MTK, 2025-01-30 (15)  DNA STRUCTURE ANTIPARALLEL DOUBLE HELIX  There are two outer sugar- phosphate backbones, with the nitrogenous bases paired in the molecule’s interior  The two strands (polynucleotides) are antiparallel, they run in opposite directions Along a length of DNA, one polynucleotide will run 5’ → 3’ and the other will be 3’ → 5’  Spacing of base-pairs is 0.34 nm DNA Structure Video: https://drive.google.com/file/d/1ZTBB9ja2yxcQQIOTcCqhLwKJypnL7ZLJ MTK, 2025-01-30 (16)  DNA STRUCTURE ANTIPARALLEL DOUBLE HELIX MTK, 2025-01-30 (17)  DNA STRUCTURE MAJOR & MINOR GROOVES  Typically, the two DNA strands wind around each other to form a right-handed helix with 10 bases per turn.  The coiling of the helix makes two grooves along the length of the molecule: Major groove – nitrogenous bases are more accessible here Minor groove – the nitrogenous bases are more occluded by the backbone How do you think this arrangement affects DNA-protein interactions? MTK, 2025-01-30 (18)  DNA STRUCTURE ALTERNATE CONFORMATIONS  Isolated DNA usually conforms to the B-DNA form: Right-handed helix Major groove is much larger than minor groove  When bound to protein, the helix budges slightly and conforms to the A-DNA form: Similar to DNA-B but less of a difference in size of grooves Wider than DNA-B  Under exotic conditions, the helix may recoil into a left- handed Z-DNA form https://onlinelibrary.wiley.com/doi/pdf/10.1002/9780470370612.ins MTK, 2025-01-30 (19)  DNA STRUCTURE ALTERNATE CONFORMATIONS A-DNA  Note the differences in the length of the molecule, the B-DNA width the of helix, and the arrangement of the base pairs Z-DNA MTK, 2025-01-30 (20)  HOW IS DNA REPLICATED?  The Watson and Crick model of the DNA double helix implies the fundamental mechanism of DNA replication: Since the two strands of DNA are complementary, each strand acts as a template for building a new strand In DNA replication, the parent molecule unwinds, and two new complementary daughter strands are built based on base-pairing rules  The experimentation that gave us our first insights into the mechanisms of DNA replication are both simple and ingenious Parent molecule Separation of strands Daughter DNA molecules A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C MTK, 2025-01-30 (21)  HOW IS DNA REPLICATED? Conservative model  Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) Semiconservative and one newly made strand model  Competing, but ultimately false, models were: Conservative model - the two parent strands rejoin Dispersive model - each strand is a mix of Dispersive old and new model MTK, 2025-01-30 (22)  HOW IS DNA REPLICATED?  Meselson and Stahl labeled the nucleotides of DNA strands with a heavy isotope of nitrogen (15N) by growing bacteria in a medium with 15N  They let the bacteria reproduce for one generation in regular media lacking this isotope – any new nucleotides would contain common 14N MTK, 2025-01-30 (23)  HOW IS DNA REPLICATED?  Their experiments supported the semiconservative model: First round of replication produced a band of hybrid DNA (all DNA contained equal amounts of 15N and 14N) - this eliminated the conservative model The second round of replication produced two discrete bands – one band that contained hybrid DNA, and another lighter band that contained DNA made with only 14N – this eliminated the dispersive model  But how exactly is DNA replicated? This experiment can’t tell us that, but stay tuned... (see Lecture 7) MTK, 2025-01-30 (24)  CHROMOSOMES  In eukaryotes, a single length of DNA found in the nucleus is called a chromosome and can be 100 millions+ base pairs long Prokaryotes have shorter, usually circular chromosomes  Chromosomes often come in pairs (homologous chromosomes) like in human somatic cells MTK, 2025-01-30 (25)  CHROMOSOMES SETS OF CHROMOSOMES IN HUMAN CELLS  In most cells of the body, humans have 23 pairs of chromosomes (46 total) One set of 23 chromosomes inherited from the mother and the other set from the father  Chromosomes of a homologous pair are the same length and shape and carry the same genes, though usually different alleles of the genes MTK, 2025-01-30 (26)  CHROMOSOMES SETS OF CHROMOSOMES IN HUMAN CELLS  A diploid cell (2n) has two sets of chromosomes  For humans, the diploid number is 46 (2n = 46)  In a cell where DNA synthesis has occurred but has not yet divided, each replicated chromosome consists of two identical sister chromatids MTK, 2025-01-30 (27)  CHROMOSOMES SETS OF CHROMOSOMES IN HUMAN CELLS  Karyotyping is often performed as a prenatal test after chorionic villus sampling (CVS) or amniocentesis  Some diseases can be diagnosed based on abnormal number of chromosomes or abnormal chromosomal structure (length, staining patterns) Why would different parts of the chromosome stain differently? MTK, 2025-01-30 (28)  CHROMOSOMES SETS OF CHROMOSOMES IN OTHER ORGANISMS  The number of chromosomes is not as important as the total information stored within them Similar species may differ radically in the number and structure of their chromosomes but possess similar genes in both number and sequence How and why would this happen in nature? MTK, 2025-01-30 (29)  DNA PACKAGING SUPERCOILING CONDENSES DNA  Bacteria have single, multiple, linear, or circular chromosomes depending on the species, but a single circular chromosome is most common  DNA bound to small amounts of histone-like protein and RNA and localized to a region of the bacterial cell called the nucleoid  The bacterial DNA is negatively supercoiled and folded into loops  Supercoiling occurs in eukaryotes, but this is not sufficient (why?) MTK, 2025-01-30 (30)  DNA PACKAGING SUPERCOILING CONDENSES DNA Supercoiling in Bacteria Video: https://drive.google.com/file/d/1Id2w82O2l82vAOkroI_rMX1dqidqpYee MTK, 2025-01-30 (31)  DNA PACKAGING DISCOVERY OF EUKARYOTIC DNA PACKAGING  Eukaryotic chromosomes are made of a combination of DNA and proteins; this combination is called chromatin  In the 1960’s, X-ray diffraction studies of revealed that chromatin has a repeating structural subunits  When isolated from cells, chromatin fibers appear as a series of tiny particles attached by thin filaments (“beads-on-a-string”)  The “beads” are called nucleosomes A core made of proteins called histones DNA wraps around histones, forming the “bead” DNA between nucleosomes lacks protein, the “string” MTK, 2025-01-30 (32)  CHROMATIN LEVELS OF PACKAGING  DNA is packaged together with histones, enabling it to be packed into dense structures, allow chromosomes to be easily separated during mitosis  What do you think is the most important chemical characteristic of histone proteins? Chromatin Packaging Video: https://drive.google.com/file/d/1blOWNmKFLzDGrpKNrooF4N_U8MF1Aul1 MTK, 2025-01-30 (33)  CHROMATIN LEVELS OF PACKAGING  2-nm fiber: bare DNA  10-nm fiber: DNA packaged into string of nucleosomes  30-nm fibers: organized, tight looping of nucleosomes  700-nm fiber: looped domains of chromatin, visible chromosomal structure MTK, 2025-01-30 (34)  CHROMATIN THE NUCLEOSOME  147 bp DNA per nucleosome (1.67 turns around the histone core)  50 bp linker between nucleosomes  5 types of histone proteins – EXTREMELY conserved between eukaryotes Why do you think this is? What charge do you think histone proteins have? MTK, 2025-01-30 (35)  CHROMATIN CHROMOSOMES DURING INTERPHASE  Chromatin changes in packaging during the cell cycle  At interphase (when the cell is not dividing), chromosomes are not highly condensed, but they still occupy specific restricted regions in the nucleus  Dense packing (called heterochromatin) makes it difficult for the cell to express genetic information coded in these regions, silencing those genes MTK, 2025-01-30 (36)  CHROMATIN CHROMOSOMES DURING INTERPHASE  The nucleolus is the most prominent part of the nucleus and is the site of ribosomal RNA (rRNA) synthesis  Multiple chromosomes contribute to the nucleolus MTK, 2025-01-30 (37)  CHROMATIN REMODELING  Chromatin can be remodeled (repackaged) to alter the accessibility of genes and plays a central role in epigenetics Epigenetics refers to the variations traits that cannot be accounted for by the sequence of the genome alone  Chromatin-remodeling complexes loosen the DNA and push it along the histone octamer MTK, 2025-01-30 (38)  CHROMATIN REMODELING HETEROCHROMATIN  Heterochromatin-specific histone modifications allow heterochromatin to form and to spread  These modifications attract heterochromatin-specific proteins that reproduce the same histone modifications on neighboring nucleosomes How does this affect gene expression in these regions? What is epigenetics?  In this manner, heterochromatin can spread until it encounters a barrier DNA sequence that blocks further propagation into regions of euchromatin MTK, 2025-01-30 (39)  CHROMATIN REMODELING HISTONE MODIFICATION  Histones can undergo chemical modifications that result in changes in chromatin organization and thus change gene expression  Each histone has a protruding tail that can be tagged by the addition of methyl, acetyl, or phosphate groups or other groups  Various combinations of these tags create a histone code MTK, 2025-01-30 (40)  CHROMATIN REMODELING X INACTIVATION IN FEMALE MAMMALS  In mammalian females, one of the two X chromosomes in most cells is randomly inactivated during embryonic development  The inactive X condenses into a Barr body, which is highly heterochromatinized  If a female is heterozygous for a particular gene located on the X chromosome, she will be a mosaic for that character (e.g., tortoiseshell and calico cats) X-inactivation Video: https://drive.google.com/file/d/1CJhWjxe4u1videQ2q_ZfsdX-gMHNzdvU MTK, 2025-01-30 (41)  EXTRA CREDIT DISCUSSION: EPIGENETICS USE BLACKBOARD DISCUSSION BOARD TO POST ANSWERS  Find a peer-reviewed research article (pubmed.gov) that deals with epigenetic influence on disease: Link to the article Give its title What disease was investigated? What kinds of chromatin modifications were observed that correlate with the disease? Do these modifications change throughout life or in response to come environmental influence? Can the modifications be inherited? MTK, 2025-01-30 (42)