Cellular and Molecular Biology Topics
Nucleic
Acids
Both DNA and RNA are linear
polymers of nucleotides. In RNA the pentose is always ribose, in DNA is always
deoxyribose. The bases for DNA are adenine, guanine, cytosine and thymine. The
bases for RNA are adenine, guanine, cytosine, and uracil. Nucleotides are linked
to each other by phosphodiester groups.
Each molecule of DNA has two strands held together by hydrogen bonding between
bases. When nucleotides polymerize, the hydroxyl group attached to the 3’ carbon
of the sugar of one molecule bonds to the phosphate of another nucleotide. The
phosphate is attached to the 5’ carbon of the next sugar. Therefore, the chain
has a 5’ to 3’ orientation because synthesis proceeds in that direction. The
3’ end of the molecule has a free hydroxyl group, and the 5’ end has a free
phosphate group. A DNA molecule has two strands running in opposite directions
(antiparallel strands).
Adenine pairs with thymine or uracil. Cytosine pairs with guanine.
Ribonucleotides have a hydroxyl group at the 2’ position of ribose, which is not present in deoxyribonucleotides. Ribonucleotides use the base uracil instead of the thymine used in deoxyribonucleotides. RNA hydrolyzes very rapidly in alkaline environment due to the hydroxyl at the 2’ position. The result is a mixture of 2’ ribonucleotide monophosphates and 3’ ribonucleotide monophosphates:
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Biochemistry
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Double Helix Foms
Virtually all double-stranded DNA in cells is in the B form: a right-handed double helix, forming a very regular molecule about the same width all the way around. The base pairs are roughly perpendicular to the axis of the helix, packed very tightly together. G-C and A-T base pairs take up essentially the same amount of space. The sugar phosphate backbone wraps around the outside of the molecule, forming major and minor grooves in the helical structure, were base pairs are exposed. Proteins recognize double stranded DNA by reaching into specific base sequences inside grooves, particularly the major grooves.
RNA cannot exist in the B form because the bulky hydroxyl groups of ribose prevent the backbone from taking a straight conformation. Instead it takes an A form, a shorter and wider cylinder were the bases are not perpendicular to the molecular axis. Groves are much shallower than in the B form. RNA-DNA hybrid molecules and double stranded RNA usually exist in the A form. Double stranded DNA may exist in the A form under special conditions.
DNA can exist in a rare Z form, a left handed helix, longer and thinner than the B form, with a zigzag backbone due to a type of purine-pyramidine sequence.
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Properties of DNA
Nucleases are enzymes that cleave phosphodiester bonds in nucleic acids. They have different specificities, i.e. each recognizes a specific sequence as substrate:
Restriction endonucleases are sequence-specific, each recognizing a particular sequence to cleave. Therefore DNA and RNA can be dissected in a reproducible manner, useful for studies that require the ability to dissect DNA by size.
DNA and RNA have a nearly uniform shape and a constant charge per unit length (-1 per phosphate along the backbone at physiological pH). Therefore they can be separated on the basis of size (length). After being cut by restriction endonucleases, gel electrophoresis is used to separate the different size fragments. As voltage is applied across a gel containing buffer the polynucleotide fragments move towards the positive electrode. Shorter fragments move further, while longer fragments are slowed down by the gel matrix.
Double-stranded nucleic acids are stabilized mostly by hydrogen bonds between base pairs, although hydrophobic and van der Waals interactions also play a role. DNA is more stable at lower temperatures and higher salt concentrations. The stability of the double-helix also depends on the proportion of GC pairs: sequences with a high proportion of GC pairs will bind more readily because that base pair is joined by 3 hydrogen bonds as opposed to the 2 bond of an AT base pair.
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DNA Denaturing and Hybridization
DNA
can be denatured by heating over a long range of temperatures above normal physiological
temperature, a process known as melting. As the temperature starts to rise,
a few base pairs are separated and if cooled down they quickly reform. At this
point, since the molecule still keeps most of its structure, the unlinked bases
are close together and can form new hydrogen bonds quickly. But as the temperature
keeps increasing and more pairs are separated, the molecule starts loosing structure
and complementary bases are not near each other any more, so they cannot quickly
reform bonds, and the strands eventually separate completely. This is a cooperative
process: the more base pairs get separated, the easier it is for others to separate.
This is illustrated by the “S” shape of the DNA melting curve. The rate-limiting
step in the renaturation of DNA is the coming together of complementary base
pairs of about a dozen nucleotides, a step known as nucleation. Once nucleation
has occurred, the pairing of the remaining bases is fast.
Denatured DNA strands can only renature if they can form a complementary double helix with antiparallel strands. However, some mismatches can be tolerated depending on the condition of hybridization. High stringency conditions include low salt and high temperatures (just below the melting point) or the presence of foramide. Under such conditions, double-helices that mismatch by more than a few percent will not renature. Under low stringency conditions (high salt, low temperature), double-helices form and remain stable even with 10-40% mismatching.
The melting temperature of DNA will depend on the stringency of the solution: low salt and foramide will increase the melting temperature. High salt will decrease the melting temperature. Experimentally, solution and temperature can be adjusted to set the criteria for the percent mismatching that will allow stable hybrids to form. The ability of DNA to renature as a hybrid can be used to identify specific fragments of DNA that are complementary to known DNA strands. This can be used to relate sequences such as gene families and homologous genes in two different species.
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Eukaryotic Chromosomes
The eukaryotic chromosome is attached to the mitotic apparatus by its centromere, and each end of the chromosome is known as a telomere. Each chromosome contains one linear DNA molecule which codes for a number of genes.
Genes contain control regions called exons and introns. Exons are the sequences ultimately present in RNA, while introns (intervening sequences or IVS) are noncoding sequences which are spliced from mRNA before transcription. Chromosomes also include intergenic regions which do not code for any RNA.
Repetitive DNA refers to many copies of related sequences per genome. Some have structural roles (telomeres and centromeres), but some have no known role. The most common are short interspersed nuclear elements (SINEs). Others are long interspersed nuclear sequences (LINEs). Both SINEs and LINEs can affect regulation (?). Other sequences called microsatellites or single tandem repeat polymorphisms (STRP) are simple repeats like GTGTGTGT.. etc, and their expansion may lead to disease. There are also middle-repetitive sequences (some special genes, some regulatory elements) and unique (single copy) sequences (i.e. most of the structural genes).
Chromatin is a complex of DNA, histone proteins and non-histone proteins. Histones are small, very basic proteins, highly conserved that organize DNA into coiled structures called nucleosomes. One fourth of the histone residues are basic (Arg and Lys). They have a central globular domain involved in protein-protein interactions that stabilize the nucleosome, and a more flexible N-terminus domain that binds to DNA. Histones are often acetylated on these N-terminus domains, which regulates how tight the nucleosomes are packed. There are 5 types of histones: H1 is a linker histone, while H2A, H2B, H3 and H4 are core histones.
In the current model of a nucleosome, 146 base pairs are wrapped in a 1.75 left-handed coil around a histone octamer which contains two of each core histones. H1 binds to the outside of the wrapped DNA. Linker DNA is unwrapped stretches of 20-80 base pairs between nucleosomes. Nucleosomes are packed into a spiral or solenoid arrangement, forming fibers 30nm in diameter.
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Telomeres
Chromosomes have structures called telomeres at the ends. Telomeres reduce recombination that would otherwise be stimulated at the ends and provide stability to the chromosome. They are long repeats of a 6 nucleotide sequence (thousands of copies) that make the 3’ strand longer and fold back on itself. The human telomere sequence is TTAGGG.
If there was no telomere, each cycle of normal DNA synthesis would leave a retrograde strand shorter since there is no way to prime the synthesis at the very end of the template strand. Progressive shortening of the chromosome will lead to lethal loss of genes over the course of several cell cycles.
Telomerases are specific enzymes that add six new nucleotides to the 3’ ends of telomeres. They are ribonucleoprotein complexes containing a small amount of RNA that serves as template for the addition of the new repeat. Progressive shortening is avoided by this addition of repeats.
Telomerases are promising targets for chemotherapy because inhibitors of the enzyme could stop cell proliferation.
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DNA Supercoiling
Supercoiled DNA is a section of the molecule were the double-helix is twisted on itself, forming whrites over twists. Linking number is the number of times the two strands make a complete 360° turn. It is also equal to the number of twists plus the number of whrites.
In
prokaryotes, DNA exists as supercoiled circles, while in eukaryotes it is a
long supercoiled molecule held by nuclear proteins at the ends. For replication
to occur, two parental DNA strands must be separated, and this cannot be accomplished
in a supercoiled molecule. To separate the strands, the number of twists and
writhes must be reduced, i.e. reduce the linking number.
Topoisomerases are enzymes that reduce the linking number by opening and resealing nicks in the DNA molecule. Topoisomerase I is present in human cells, and has a tyrosine which acts as the active group to catalyze the transesterification of a phosphodiester bond. The hydroxyl in Tyr attacks the phosphate and binds covalently with it temporarily, allowing one strand to pass around the other. This reaction changes the linking number by one.
DNA gyrase (topoisomerase II) is present in E. coli, and changes the linking number by two. It is critical in E. coli, were topoisomerase I cannot reduce the linking number due to the circular nature of the bacterial DNA. Antibiotics that target DNA gyrase rapidly stop E. coli replication.
There are two modes of targeting topoisomerases. Topoisomerase inhibitors prevent catalytic activity. Topoisomerase poisons freeze the covalent DNA-protein links. Coumermycin A1 and novobiocin are inhibitors. Naladixic acid and cuprofloxacin are poisons.
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