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.

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

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

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

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

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