DNA metabolism and organization
-
Upload
medico-legal-institute -
Category
Technology
-
view
744 -
download
2
description
Transcript of DNA metabolism and organization
![Page 1: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/1.jpg)
◦ DNA replication◦ DNA repair◦ DNA Organization
Key topics:
![Page 2: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/2.jpg)
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
![Page 3: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/3.jpg)
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
![Page 4: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/4.jpg)
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
![Page 5: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/5.jpg)
Both strands are replicated simultaneously
![Page 6: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/6.jpg)
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
![Page 7: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/7.jpg)
![Page 8: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/8.jpg)
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
![Page 9: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/9.jpg)
Initiation◦ Requires initiator proteins (trans-acting factors)
Elongation◦ Leading and Lagging strands (repeated priming)
Termination◦ Circular and linear chromosomes have unique
problems
![Page 10: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/10.jpg)
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
![Page 11: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/11.jpg)
![Page 12: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/12.jpg)
![Page 13: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/13.jpg)
![Page 14: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/14.jpg)
![Page 15: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/15.jpg)
![Page 16: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/16.jpg)
![Page 17: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/17.jpg)
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
![Page 18: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/18.jpg)
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
![Page 19: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/19.jpg)
![Page 20: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/20.jpg)
![Page 21: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/21.jpg)
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
![Page 22: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/22.jpg)
•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
![Page 23: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/23.jpg)
•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
![Page 24: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/24.jpg)
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
![Page 25: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/25.jpg)
• 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)
![Page 26: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/26.jpg)
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
![Page 27: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/27.jpg)
• 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)
![Page 28: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/28.jpg)
Designations of Designations of the bands and the bands and interbands in the interbands in the human karyotypehuman karyotype
G Banding Pattern
![Page 30: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/30.jpg)
![Page 31: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/31.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
![Page 32: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/32.jpg)
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
![Page 33: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/33.jpg)
• 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
![Page 34: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/34.jpg)
HISTONES
•Small basic proteins
•Constant amount in cells
•25% lysine & arginine (Net + charge)
•5 main types: H1, H2A, H2B, H3 & H4
![Page 35: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/35.jpg)
•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)
![Page 36: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/36.jpg)
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
![Page 37: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/37.jpg)
•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
![Page 38: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/38.jpg)
•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)
![Page 39: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/39.jpg)
Nucleosome StructureNucleosome Structure
![Page 40: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/40.jpg)
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
![Page 41: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/41.jpg)
Packaging of nucleosomes into the 30-nm Packaging of nucleosomes into the 30-nm chromatin fiberchromatin fiber
![Page 42: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/42.jpg)
![Page 43: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/43.jpg)
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)
![Page 44: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/44.jpg)
![Page 45: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/45.jpg)
• 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
![Page 46: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/46.jpg)
• 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
![Page 47: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/47.jpg)
The CentromereThe Centromere
![Page 48: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/48.jpg)
• 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
![Page 49: DNA metabolism and organization](https://reader033.fdocuments.us/reader033/viewer/2022061118/545cbd30af7959b4098b4925/html5/thumbnails/49.jpg)
Centromere and TelomeresCentromere and Telomeres