Welcome Each of You to My Molecular Biology Class

Molecular Biology of the Gene, 5/E --- Watson et al. (2004) Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 3/011/05

Part II: Maintenance of the Genome Dedicated to the structure of DNA and the processes that propagate, maintain and alter it from one cell generation to the next

Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the molecular level Ch 11: Site-specific recombination and transposition of DNA 3/11/05

CHAPTER 7: Chromosomes, chromatin, and the nucleosome

Consider the structure of DNA within the cell, and the biological relevance of the structure.

Nucleus: 细胞核; Nucleolus: 核仁 Nucleoid: 类核 Mitosis: 有丝分裂；Meiosis：减数分裂 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 Histone: 组蛋白；Nucleosome: 核小体 Chromotasome: 染色小体 Chromosome: 染色体； Chromatin: 染色质；eu-; hetero- Centromere (中心粒） Telomere（端粒） Repetitive DNA (重复DNA) Tandem gene cluster（串联基因 簇）

OUTLINE Chromosome sequence & diversity Chromosome duplication & segregation The nucleosome Higher-order chromatin structure Regulation of chromatin structure Nucleosome assembly

Prokaryotic chromosome Prokaryotic and eukaryotic chromosome structure D1 Prokaryotic chromosome (原核 染色体)

D1-1 The E. coli chromosome D1 Prokaryotic chromosome structure D1-1 The E. coli chromosome A single closed-circular DNA, 4.6 X 106bp The DNA packaged into a region known as Nucleoid (类核) that contains high concentration of DNA (up to 30-50 mg/ml) as well as all proteins associated with DNA. Continuous replication (no cell cycle) Attach to a part of cell membrane by ?

– Bacterial chromosome 细菌染色体 D1 Prokaryotic chromosome structure Nucleoid (类核,拟核) – Bacterial chromosome 细菌染色体

D1-2 DNA domains/loops Observed under electron microscope D1 Prokaryotic chromosome structure D1-2 DNA domains/loops Observed under electron microscope 50-100 domains or loops per E. coli chromosome, with 50-100 kb/loop The ends of loops are constrained (束缚) by Are loops dynamic or static? What do you think, why? Could you design experiments to prove it?

Domain/ Loop Basic protein Supercoiled DNA Member binding proteins? D1 Prokaryotic chromosome structure Domain/ Loop Basic protein Member binding proteins? Supercoiled DNA

Non-supercoiled domain D4 Genome Complexity D1 Prokaryotic chromosome structure Supercoiled domain Non-supercoiled domain Protein-membrane core or scaffold

D1-3 Supercoiling of the genome D1 Prokaryotic chromosome structure D1-3 Supercoiling of the genome E. coli chromosome as a whole is negatively supercoiled Individual domains may be supercoiled independently (topological independent) because the protein-membrane scaffold may prevent DNA rotation. Oops！Direct biochemical evidence is lacking for different level of supercoiling in different domains. Do you want to try an experiment? back

D1-4 DNA-binding proteins D1 Prokaryotic chromosome structure D1-4 DNA-binding proteins back Histone-like proteins essential for DNA packaging to stabilize and constrain the supercoiling. HU: a small basic dimeric (碱性双体) protein, non-specific binding to DNA, most abundant. H-NS (protein H1): neutral monomeric (中性单体), partially non-specific binding Site-specific DNA binding proteins important for organization of DNA domains (RNA polymerases, IHF etc).

circular, multiple copies Supplementary 1: Organelle genomes: circular, multiple copies ctDNA: chloroplast (叶绿体) DNA, 140kb in plants, and <200kb in lower eukaryotes. Similar to cyanobacteria (兰细菌) mtDNA: mitochondrial (线粒体) DNA, 16.5 kb in mammals, 80 kb in yeast, >100kb in plant. Similar to -purple bacteria, Rickettsia) mtDNA

Range of genome size found in different organism phyla. Supplementary 2: Range of genome size found in different organism phyla.

D2. Chromatin Structure (染色质结构) Prokaryotic and eukaryotic chromosome structure D2. Chromatin Structure (染色质结构)

D2 Chromatin structure D2-1. Chromatin (染色质) A highly organized complex of DNA and protein (nucleoprotein complex), which makes up the eukaryotic chromosomes (染色体). >50% of the mass is protein Solving the packing problem: chromatin The length of a chromosomal DNA can be up to several centimeters (cm), but the the diameter of nucleus is about 1-10 mm. (104-fold of condensation) The DNA concentration in nucleus is about 200 mg/ml

D2 Chromatin structure Chromatin structure enables the chromosomes to alter their compactness as the cell progress the cell cycle. Mitosis Interphase Chromatin (diffused) Chromosome (condensed)

D2-2&3. Histones (组蛋白) and Nucleosomes (核小体) D2 Chromatin structure D2-2&3. Histones (组蛋白) and Nucleosomes (核小体) The major protein components of chromatin Four families of core histone: H2A, H2B, H3 and H4. An additional non-core histone H1 Small, 10 kDa for core histones and 23 kDa for H1. Basic (rich in lysine and arginine) and tightly binds to DNA

D2 Chromatin structure “Members of the same histone class are very highly conserved between unrelated species, but there is not much similarity in sequence between the different histone class (page 50 of your text book). ” What does this fact suggest to you?

Histone octamer (组蛋白八聚体) D2 Chromatin structure Histone octamer (组蛋白八聚体) Top view Nucleosome core Side view

Nucleosome core 146 bp, 1.8 superhelical turn D2 Chromatin structure DNA Histone octamer Nucleosome core 146 bp, 1.8 superhelical turn Histone H1 Chromatosome 166 bp, 2 superhelical turn

D2 Chromatin structure D2-4. The role of H1 Stabilizes the point at which DNA enters and leaves the nucleosome core. C- tail of H1: stabilizes the DNA between the nucleosome cores. 23 kDa, located outside of nucleosome core, binds to DNA more loosely Less conserved in its sequence (What does this suggests?)

D2 Chromatin structure D2-5. Linker DNA The additional DNA required to make up the 200 bp nucleosomal repeat, ~55 bp How was the linker identified?

“Beads on a string” structure Nucleosome repeat: Core + linker DNA 200 bp Histone H1 Nucleosome Linker DNA <10 to > 100 bp average 55 bp

D2-6. The 30 nm fiber - Steps to make it D2 Chromatin structure D2-6. The 30 nm fiber - Steps to make it

Step 1: form a Nucleosome (核小体) D2 Chromatin structure Step 1: form a Nucleosome (核小体) DNA + Histone octamer (组蛋白八聚体) Nucleosome core (核小体核心 146bp) + H1  Chromatosome (染色小体 166bp) + linker DNA  Nucleosome (核小体) (~200 bp of DNA) Nucleosome (核小体) is the basic structural subunit of chromatin, consisting of ~200 bp of DNA and an octamer of histone proteins.

“Beads on a string” structure D2 Chromatin structure Step 2: “Beads on a string” structure Nucleosome repeat: Core + linker DNA 200 bp Histone H1 Nucleosome Linker DNA <10 to > 100 bp average 55 bp

The 30 nm fiber Step 3: 30 nm fiber Higher ordered Left-handed helix D2 Chromatin structure The 30 nm fiber 30 nm fiber (30nm in diameter) Solenoid (螺线管） Step 3: 30 nm fiber Higher ordered Left-handed helix Six nucloesomes per turn

Step 4: looped domain structure D2 Chromatin structure Step 4: looped domain structure Highest level of chromatin organization 30 nm fiber 300 nm Nuclear matrix (核基质), protein complex

Nucleosomes and micrococcal nuclease treatment Supplementary 1: Nucleosomes and micrococcal nuclease treatment

Steps from DNA to chromosome Supplementary 2: Steps from DNA to chromosome

D3 Eukaryotic chromosome (真核染色体) Prokaryotic and eukaryotic chromosome structure D3 Eukaryotic chromosome (真核染色体)

D3-1 Cell cycle (细胞周期) (增补内容) Prokaryotic and eukaryotic chromosome structure D3-1 Cell cycle (细胞周期) (增补内容)

Cell cycle Interphase 间期: G1 + S + G2 (G0) M phase (mitosis 有丝分裂): D3 Eukaryotic chromosome structure Cell cycle Interphase 间期: G1 + S + G2 (G0) M phase (mitosis 有丝分裂):

D3 Eukaryotic chromosome structure D3 Eukaryotic Chromosomal Structure Cell cycle M phase includes: prophase (前期), metaphase (中期), anaphase (后期), telophase (末期)

D3 Eukaryotic chromosome structure Mitotic chromosome

more condensed than chromatin, and most highly condensed at mitosis D3 Eukaryotic chromosome structure D3-2 Mitotic chromosome (有丝分裂期的染色体) more condensed than chromatin, and most highly condensed at mitosis

Mitotic chromosome at metaphase D3 Eukaryotic chromosome structure Telomere Sister chromatid 姊妹染色单体 Centromere 中心粒 Mitotic chromosome at metaphase Nuclear matrix 核基质 Loops of 30nm fiber Chromatid 染色单体

The centromere (中心粒) Yeast: D3 Eukaryotic chromosome structure The centromere (中心粒) The region where two chromatids (姊妹染色体）are joined The sites of attachment to the mitotic spindle (纺锤体) via kinetochore (动原体) Centromere DNA: Yeast: AT-rich (88bp)

Mitotic chromosome D3 Eukaryotic chromosome structure Mitotic spindle D3 Eukaryotic Chromosomal Structure Mitotic chromosome Mitotic spindle

Mammalian cells: much longer, flanked by satellite DNA (卫星DNA) D3 Eukaryotic chromosome structure Mitotic chromosome - centromere Yeast centromere Mammalian cells: much longer, flanked by satellite DNA (卫星DNA)

D3 Eukaryotic chromosome structure The Telomere (端粒) Specialized DNA sequences which form the ends of the linear DNA of the eukaryotic chromosome Contains up to hundreds copies of a short repeated sequence (5’-TTAGGG-3’ in human) Synthesized by the enzyme telomerase (a ribonucleoprotein) independent of normal DNA replication. The telomeric DNA forms a special secondary structure to protect the chromosomal ends from degradation

Telomere & Telomerase D3 Eukaryotic chromosome structure D3 Eukaryotic Chromosomal Structure Telomere & Telomerase Repeat sequence: Tetrahymena- TTGGGG; human- TTAGGG

A loop structure forms at the end of chromosomal DNA D3 Eukaryotic chromosome structure Telomere: structure A loop structure forms at the end of chromosomal DNA

D3-3 Interphase chromosomes Prokaryotic and eukaryotic chromosome structure D3-3 Interphase chromosomes (间期的染色体结构)

Interphase chromosomes: chromatin (染色质) D3 Eukaryotic chromosome structure Interphase chromosomes: chromatin (染色质) Heterochromatin (异染色质) Highly condensed Transcriptionally inactive Can be the repeated satellite DNA close to the centromeres, and sometimes a whole chromosome (e.g. one X chromosome in mammals)

Euchromatin (常染色质) : chromatin other than heterochromatin. D3 Eukaryotic chromosome structure Euchromatin (常染色质) : chromatin other than heterochromatin. More diffused and not visible The region where transcription takes place Not homogenous, only a portion (~10%) euchromatin is transcriptionally active where the 30nm fiber has been dissociated to “beads on a string” structure and parts of these regions may be depleted of nucleosome.

D3 Eukaryotic chromosome structure DNase I hypersensitivity: How to map the regions of transcriptionally active chromaatin ?

Euchromatin CpG methylation (甲基化): CpG island (CpG 岛) D3 Eukaryotic chromosome structure Euchromatin CpG methylation (甲基化): CpG island (CpG 岛) Methylation of C-5 in the cytosine base of 5’-CG-3’ Occurs in mammalian cells Signaling the appropriate level of chromosomal packing at the sites of expressed genes CpG methylation is associated with transcriptionally inactive regions of chromatin Islands of unmethylated CpG are coincident with regions of DNase I hypersensitivity “Islands”: surround the promoters of housekeeping genes. Responsible for epigenetic (表位遗传）and may also to RNA silencing (RNA 沉默)

D3 Eukaryotic chromosome structure Euchromatin

Fig. 3. CpG islands and the promoters of housekeeping genes D3 Eukaryotic chromosome structure Fig. 3. CpG islands and the promoters of housekeeping genes

Transcription & nucleosome formation Supplementary Transcription & nucleosome formation

D3-4 Histone variants and modification Prokaryotic and eukaryotic chromosome structure D3-4 Histone variants and modification (组蛋白的变体和修饰） The major mechanisms for the condensing and decondensing of chromatin operate directly through the histone proteins which carry out the packaging.

D3 Eukaryotic chromosome structure Short-term changes in chromosome packing modulated by chemical modification of histone proteins Actively transcribed chromatin: via acetylation (乙酰化) of lysine residues in the N-terminal regions of the core histones. Condensation of chromosomes at mitosis: by the phosphorylation (磷酸化) of histone H1.

D3 Eukaryotic chromosome structure Longer term differences in chromatin condensation: associated with changes due to stages in development (发育) and different tissue types. Utilization of alternative histone variants, H5 replacing H1 in some very inactive chromatin.

D4 Genome Complexity (基因组的复杂性) Prokaryotic and eukaryotic chromosome structure D4 Genome Complexity (基因组的复杂性)

genetic complexity (遗传复杂性) D4 Genome Complexity D4-1 Genome (基因组) & genetic complexity (遗传复杂性) (增补内容)

Range of genome size in different phyla D4 Genome Complexity Range of genome size in different phyla 植物 动物 真菌 等 细菌

Genome & genes (基因组和基因的关系） Genome: all DNA sequences in a cell D4 Genome Complexity Genome & genes (基因组和基因的关系） Genome: all DNA sequences in a cell Genes: a stretch of continuous DNA sequence encoding a protein or RNA C-value is the quantity of DNA in the genome (per haploid set of chromosomes). C-value paradox (C值矛盾) refers to the lack of a correlation between genome size and genetic complexity

D4-2 Reassociation kinetics D4 Genome Complexity D4-2 Reassociation kinetics (重新结合动力学) Genomic DNA extraction  Sonication or shearing to a uniform size (x 100-1000 bp)  3. Thermal Denaturation  4. Re-annealing 5. Measure & plotting the re-annealing process will yield kinetics

moderately repetitive DNA D4 Genome Complexity Highly repetitive DNA moderately repetitive DNA unique DNA Human E. coli Measuring methods： Spectroscopy/UV absorption hydroxyapatite (羟[基]磷灰石)chromotography

D4-3: non-coding sequence D4 Genome Complexity D4-3: non-coding sequence DNA sequence that does not code for protein or RNA, including Introns (unique sequence) in genes DNA consisting of multiple repeats, can be tandemly repeated sequences (串联重复序列）(e.g. satellite DNA) or interspersed repeats (分散重复) (e.g. Alu element) etc.

D4-4 Unique sequence DNA (单一序列DNA) The slowest to reassociate D4 Genome Complexity D4-4 Unique sequence DNA (单一序列DNA) The slowest to reassociate Corresponds to coding regions of genes occurring in one or a few copies/haploid genome All the DNA in E. coli genome has a unique sequence. Why E. coli DNA associate fast?

D4-5 Repetitive DNA (重复序列DNA) D4 Genome Complexity D4-5 Repetitive DNA (重复序列DNA) Tandem gene clusters (串联基因簇）: (1) moderately repetitive DNA consists of a number of types of repeated sequence. (2) genes whose products are required in unusually large quantities, e.g. there are 10-10000 copies of rDNA encoding 45S precursor and X100 copies of histone genes.

Alu + L1= ~ 10% of human genome. D4 Genome Complexity Dispersed repetitive DNA (离散重复序列） Moderately repetitive (x100- x1000 copies) Scattered throughout the genome Human Alu elements: 300bp, 300 000 –500 000 copies of 80-90% identity Human L1 element Alu + L1= ~ 10% of human genome. Functions of these repetitive DNA：largely unknown

Satellite DNA (卫星DNA, simple sequence) : D4 Genome Complexity Satellite DNA (卫星DNA, simple sequence) : Highly repetitive DNA (>106). very short (2 to 20-30bp, mini- or micro-), in tandem arrays concentrated near the centromeres and forms a large part of heterochromatin. as separate band in buoyant density gradient no function found, except a possible role in kinetochore binding Minisatellite repeats are the basis of the DNA fingerprinting techniques (指纹印迹), Why?

Satellite DNA Mouse genome DNA 30% GC in satellite DNA D4 Genome Complexity Satellite DNA Mouse genome DNA 30% GC in satellite DNA CsCl centrifugation

Drosophila satellite DNA repeat (several million copies) D4 Genome Complexity 5’ – ATAAACTATAAACTATAAACT – 3’ 3’ – TATTTGATATTTGATATTTGA – 5’ n ACAAACT, 1.1x107 bp, 25% genome ATAAACT, 3.6x106 bp, 8% genome ACAAATT, 3.6x106 bp, 8% genome AATATAG, cryptic Satellites comprise more than 40% of the genome Drosophila satellite DNA repeat (several million copies)

Genes in Drosophila genome Supplementary Genes in Drosophila genome

Summary Prokaryotic chromosome: closed-circular DNA, domains/loops, negatively supercoiled, HU & H-NS Eukaryotic chromatin: Histones (octamer: H2A, H2B, H3, H4)+146bp DNA > Nucleosomes + H1 > chromatosome + Linker DNA > beads on string > 30nm fiber > fiber loop + nuclear matrix > highly ordered chromatin > > > chromosome Eukaryotic chromosome structure: centromere, kinetochore, telomere, hetero- or euchromatin, CpG island and methylation Genome complexity: noncoding DNA, unique sequence, repetitive DNA, satellite DNA

Homework (on the CD) See the animations for DNA topology, Topoisomerase, as well as Ribozyme Structure and Activity. Answering the questions in “applying your knowledge” is required. Play the structural tutorial “Introduction to the DNA structure” to better understand DNA structure Finish all the critical thinking exercise