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Oct 2009

LSM 1101 Biochemistry of Biomolecules Dr. Swami’s class notes Class 1a: Nucleic acids Our class on DNA is divided into 3 parts: (I) Genetics (II) DNA structure (III) Concepts and applications. I. Genetics: In the primordial period, simple molecules were formed from atoms and from these molecules, macromolecules were formed. These macromolecules formed life and all living organisms. The classical genetic and heredity observations in the 19th century started the search for the origin of life. The transforming principle of DNA was demonstrated from the experiment in which non-pathogenic (R-form) and virulent (S-form) but heat treated bacteria, when co-injected, could kill the mice. After that, the link between genes (DNA) and genotype / phenotype was established. II. DNA structure: The genomic DNA of a eukaryotic cell is located in a special organelle, the nucleus, whereas in a prokaryotic cell there is no nucleus. In a virus, including bacteriohage, the genome is packed efficiently. In a human cell, the complete genetic DNA is organized into 23 pairs of chromosomes. Chromatid is one of the two identical copies of DNA in a chromosome. The two copies approach each other at the centromere. The ends of DNA in a chromosome are called telomere. The location of a gene in a chromosome is marked as, say, 7q31.2 where 7 refers to the chromosome number, q is the long arm (the short arm of the chromosome is called ‘p’), 3 refers to the region of a chromosome when colored using a particular process, 1 refers to band 1 in that region and 2 refers to a sub-band within band 1. In the chromatin, DNA is wound around the histone core (made by 2 copies each of the H2A, H2B, H3 and H4 proteins) and clamped by the H1 protein. Anytime this DNA is accessed for any biochemical reaction, there will be physical rearrangement of DNA and the histone core and furthermore the histone proteins undergo chemical modifications, like acetylation and methylation. Two strands of DNA form duplex DNA through base-pairing. In a basepair, the two bases are unlikely to be perfectly aligned or coplanar. In the same token, two adjacent basepairs also need not be perfectly parallel to each other. There are three forms of DNA: B-DNA, A-DNA and Z-DNA. The B form is the physiological form. The other two forms are man-made from specific sequences. While the first two forms are right handed helices, the last one is left-handed. In the B-form, the minor groove is narrow and the major groove is wide whereas in the A and Z forms, the groove widths are nearly the same. Also, a basepair in the B-form cuts the helical axis whereas in the A-form, a basepair is very much away from the helical axis. However, in the Z-form a basepair lies in-between.


transcription (the making of mRNA using a DNA template) and translation (the making of a protein using mRNA as a template). the new circular adjusts (writhes) with two cross-overs. which is also 26. That means. RNAs are also very important in several cellular processes. displaying its amino acid acceptor region and the anticodon region. There are 3 types of RNA. DNA exists in a supercoiled form. Fig. If you over-wind by two. 2 . DNA is unwound by enzymes like helicases and long leading strands ( for the parental 3’ to 5’ strand) and several short lagging strands (for the parental 5’ to 3’ strand) are made by the DNA polymerase. L = 28 and W = +2. start from base-pair 1 on a strand and come to the same but one earlier position on the same strand after 10 base-pairs. 1. The short fragments are joined by L = T + W. In prokaryotic DNA replication. Now. the important fundamental processes are: the cell cycle (including DNA replication – the making of DNA using a DNA template). mRNA. DNA replication: In molecular biology. The twist T = total base-pairs / 10 = 260/10 = 26. or 26 = 26 + 0 Now cut only one strand and unwind that strand two times and reconnect the ends. An amino-acyl tRNA synthetase enzyme attaches a corresponding amino acid to the tRNA. the tRNA is normally depicted in the ‘clover leaf’ form. rRNA and tRNA. 10 9 8 7 6 5 4 3 2 1 Strand-1 Strand-2 Fig. we will learn only a few applications. Several studies have established the connection between the number of base-pairs (linking number. due to limitation of time. L becomes 24. the next 10 base-pairs form the next one round and so on). the circular DNA writhes by 2 but in the opposite direction. 1. Class 1b III. The making of a protein using a DNA template is not yet known. Or. However. 1. 24 = 26 – 2 or W becomes -2. Even now.Supercoiled DNA: In a chromosome (or even in a circular plasmid). Of these 3 classes. In order to balance the above equation. B-DNA Apart from DNA. twist) and the level of supercoiling (writhing number). Applications and concepts: There are several applications and processes that involve nucleic acids. So the equation becomes. The next level of events includes reverse transcription (the making of DNA using an RNA template) and the making of RNA using an RNA template. The linking number is the number of times one strand crosses the other. Assume there are 260 B-DNA base-pairs (10 base-pairs will form one full turn. convert the linear DNA into circular DNA by connecting the ends of the same strands.

as a dimer. This way. the mini chromosome maintenance proteins 2 to 7 (MCM 2-7) bind to the above proteins. Make complementary DNA for all the RNAs in the cells. Then MutL binds. An endonuclease is recruited to cut the mismatch site and DNA is removed from the region. 3. DNA replication must be initiated only once per origin per cell cycle. In a prokaryotic cell. a cancer cell) behave and what are the genes that are upregulated and down-regulated. even geminin is degraded. Geminin binds to Cdt1 and primes it for degradation. In a eukaryotic cell. selected enzymes fix those problems. The process of mistmatch repair is almost the same in a eukaryotic cell. if there is a base-pair mismatch. the genes that are active only in the cancer cell will bind to their complementary fragments and will emit red signal. Next. We are not going to review that mode here. One such repair is DNA mismatch repair. DNA repair: Several types of problems happen during DNA synthesis. there cannot be another DNA replication firing. From this we can learn which genes are upregulated and down regulated in a particular cell for a particular disease condition. Now pass the two pools of DNA through the chip. origin replication protein complex (ORC) binds to the origin of replication. Once DNA replication is initiated. DNA replication takes place only once per cell cycle. a protein. Take a normal cell and a cancer cell.ligases. 2. The MutS protein. during DNA synthesis. immediately DNA synthesis is halted. The other mode is through the involvement of geminin. the nucleus of an egg is removed and a nucleus from any suitable cell from an individual is implanted. 3 . On a commercial DNA chip. We have published the structure of geminin. The DNA polymerase enzyme remakes the stretch again without any mistake. But in cloning. At the end of the cell cycle. Treat the normal cell DNA with a dye (say green) and that of the cancer cell with a red dye. Similarly. The assembly of all these proteins is called ‘licensing’ and the above complex of all these proteins is called the pre replication complex (pre-RC). This cell grows with the same genetic make-up of the nucleus donor (not the egg donor). These proteins pull the sector where the mismatch is present. there are several origins of DNA replication (dedicated sequences in DNA) in a chromosome. Next. The CDT1 protein binds to CDC6. The geminin-Cdt1 complex structure is also published by another group. Once Cdt1 is removed from the pre-RC. The first mode is through the involvement of cyclin dependent kinases (CDKs). unique and short single stranded DNA fragments of all known human genes (as of today) are immobilized on glass. If there is any problem during DNA synthesis. Cloning: In conventional sexual reproduction or in vitro fertilization (IVF). DNA microarray: This development is an important tool to study how a normal cell and an affected cell (say. There are two modes by which DNA re-replication is prevented. first binds at the mismatch site. an egg is impregnated by a sperm cell. MutH binds and form the end clamp. The CDC6 protein (CDC28 in yeast) binds to ORC. First. The genes that are active only in the normal cell (thereby making mRNA and hence cDNA) will bind to their complementary fragments (immobilized on the chip) and will emit green signal when detected. like base-pair mismatch. The genes that are common to both cells will give out yellow signal.

especially in the promoter region. using a modified virus. amino-acyl tRNA synthetases bind to tRNAs and attach corresponding amino acids to them. the gene of good insulin is first inserted into a suitable vector with a promoter. This construct is delivered to the organ of expression. The normal mode of treatment is administering these proteins by injection. Similarly. can be used as reporters to identify the location of protein expression for a protein of interest. and then looks for any messenger RNA that matches it. Researchers use these small RNA molecules to fight disease. MBD 1-4) bind to these methylated regions of the promoter and do not allow any access to RNA polymerase. form long stretches of double-stranded RNA when they replicate.4. When our cells find double-stranded RNA. the gene of Factor VIII is delivered to the liver. it is often a sign of an infection. Transgenic / reporter genes: Selected color displaying proteins. called Methylated DNA Binding Domain (MBD) proteisn (MeCP2. called the CG islands. tRNAs and selected RNA regions are double-stranded. which is used as a delivering agent. 7. it cleaves the RNA. thereby preventing transcription of the gene. we can synthesize a non-natural interfering RNA. 5. Furthermore. say pancreas. termed RNA interference. 6. CG methylation can be studied using a technique called pyrosequencing. However. in a sequence seems to be very random. CpG methylation and gene silencing: The studies on how a tumor suppressor gene is silenced in a cancer cell led to the discovery of CG methylation and gene silencing. CGs occur more frequently. Such techniques can be used to generate multicolored ornamental fish for the same species. followed by G. These genes express the corresponding proteins at the corresponding organ. occur because of the lack of related proteins. For example. using them to knock out cancer genes. Based on this principle. transcription factors bind to the promoter / enhancer regions of a gene. The GFP gene is attached to the gene of our interest and injected in an embryo and the location of protein expression is visually observed. called small interfering RNA (SiRNA) by the protein Dicer. RNA interference: Most of the free forms of RNA. If it finds some. in gene therapy. then insert it into a cell to destroy any messenger RNA that we desire. DNA polymerase is involved in DNA replication and RNA polymerase is important for transcription. destroying it. A set of proteins. however. like green fluorescent protein (GFP). In this way. Gene therapy: Certain diseases. The argonaute protein strips away one strand from the siRNA. Viral double-stranded RNA are cut into pieces (about 21 base-pairs). DNA protein interaction: Several proteins interact with DNA. Statistically. plant and animal cells have a more targeted defense that attacks the viral double stranded RNA directly. The pattern of CG methylation in a normal cell and cancer cell can identify the level of gene silencing (and hence the indirect clues on the level of disease progress and treatment). the occurrence of C. However. However. for instance. 4 . in most of the genes. messenger RNA molecules in particular. are single strands. the cell removes any messenger RNA that is the same as the original double-stranded piece found and processed by dicer. Restriction enzymes bind to and cut DNA. in the case of diabetes. Many viruses. like insulin dependent diabetes and hemophilia. The C of these CG bases get methylated.

Another example is PARN. uridine is modified to pseudo-uridine by pseudo-uridine synthase enzymes. which truncates the poly-A tail of mRNAS. RNA modifying enzymes: RNA has to be modified in selected cellular processes. For example.8. 5 .