DNA Replication

Topoisomerase It wraps around DNA and makes a cut permitting the helix to spin. Once DNA is relaxed, topoisomerase reconnects broken strand. Topoisomerase (type I &type II) is an isomerase enzyme

Helicases Are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e. DNA, RNA, or RNA-DNA hybrid) using energy derived from nucleotide hydrolysis.

Primase A primer is a short RNA segment that is complementary to a DNA segment, and is necessary to begin DNA replication. Primase is of key importance in DNA replication because no known DNA polymerases can initiate the synthesis of a DNA strand without initial RNA primers.

leading strand is the DNA strand at the opposite side of the replication fork from the lagging strand. It goes from a 5` - 3` direction (these numbers indicate the position of the molecule in respect to the carbon atoms it contains).

lagging strand In DNA replication, the lagging strand is the DNA strand at the opposite side of the replication fork from the leading strand. It goes from 3' to 5' (these numbers indicate the position of the molecule in respect to the carbon atoms it contains). On the lagging strand, primase "reads" the DNA and adds RNA to it in short bursts. Pol III lengthens the bursts, forming Okazaki fragments. Pol I then "reads" the fragments, removes the RNA using its flap endonuclease domain, and adds its own nucleotides (this is necessary because RNA and DNA use slightly different kinds of nucleotides). DNA ligase joins the fragments together.

Okazaki fragment When the lagging strand is being replicated on the original strand, the 5'-3' pattern must be used; thus a small discontinuity occurs and an Okazaki Fragment forms. These fragments are processed by the replication machinery to produce a continuous strand of DNA and hence a complete daughter DNA helix.

DNA & RNA Polymerases A polymerase is an enzyme whose central function is associated with polymers of nucleic acids such as RNA and DNA. The most well-known function of a polymerase is the catalysis of production of new DNA or RNA from an existing DNA or RNA template in the processes of replication and transcription

DNA polymerase A DNA polymerase is an enzyme that assists in DNA replication. Such enzymes catalyze the polymerization of deoxyribonucleotides alongside a DNA strand, which they "read" and use as a template. The newly-polymerized molecule is complementary to the template strand and identical to the template's partner strand. DNA-Polymerase initiates DNA replication by binding to a piece of single-stranded DNA.

DNA polymerase Prokaryotic DNA polymerases (5 pol.) Pol I: implicated in DNA repair Pol II: involved in replication of damaged DNA Pol III: the main polymerase in bacteria Pol IV& Pol V: participates in bypassing DNA damage. Eukaryotic DNA polymerases Pol α: acts as a primase (synthesizing a RNA primer), and then as a DNA Pol elongating that primer with DNA nucleotides. Pol β: is implicated in repairing DNA. Pol γ: replicates mitochondrial DNA. Pol δ: is the main polymerase in eukaryotes Pol ε: uncertain. η, ι, κ, and ζ involved in the bypass of DNA damage. There are also other eukaryotic polymerases known, which are not as well characterized: θ, λ, φ, σ, and μ.

RNA polymerase RNA polymerase (RNAP or RNApol) is an enzyme that makes an RNA copy of a DNA or RNA template. In cells, RNAP is needed for constructing RNA chains from DNA genes, a process called transcription. RNA polymerase enzymes are essential to life and are found in all organisms and many viruses. In chemical terms, RNAP is a nucleotidyl transferase that polymerizes ribonucleotides at the 3' end of an RNA transcript.

RNA polymerase Prokaryotic RNA polymerase α2: the two α subunits assemble the enzyme and recognize regulatory factors. β: this has the polymerase activity (catalyzes the synthesis of RNA) which includes chain initiation and elongation. β': binds to DNA (nonspecifically). ω: restores denatured RNA polymerase to its functional form in vitro. Eukaryotic RNA polymerase RNAp I synthesizes a pre- rRNA, which matures into rRNA which will form the major RNA sections of the ribosome. RNAp II synthesizes precursors of mRNA RNA p III synthesizes tRNAs, rRNA and other small RNAs found in the nucleus and cytosol.

Wobble base pair A wobble base pair is a G-U and I-U / I-A / I-C pair fundamental in RNA secondary structure. Its thermodynamic stability is comparable to that of the Watson-Crick base pair. Wobble base pairs are critical for the proper translation of the genetic code. The genetic code makes up for disparities in the number of amino acids (20) for codons (64), by using modified base pairs in the first base of the anti- codon. One important modified base is inosine which can pair with three bases: uracil, adenine, and cytosine. Another critical base pair is the G-U base pair, which allows uracil to pair with two bases: guanine and adenine.

Wobble base pairs for inosine Wobble base pairs for Uracil

DNA Cloning A plasmid is a DNA molecule separate from the chromosomal DNA and capable of autonomous replication. It is typically circular and double- stranded. It usually occurs in bacteria, sometimes in eukaryotic organisms. Size of plasmids varies from 1 to over 400 kilobase pairs (kbp). There may be one copy, for large plasmids, to hundreds of copies of the same plasmid in a single cell, or even thousands of copies, for certain artificial plasmids selected for high copy number. Plasmids can be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.

A cosmid, is a type of plasmid, constructed by the insertion of cos sequences, Cos sequences are single stranded sequences of DNA, which have been split from the parent molecule by a specific restriction enzyme in such a way that the ends have specific affinity for each other, and hence are known as cohesive ends. Cosmids are packaged in phage structures consisting of proteins, which allows the foreign genes to be inserted into the bacteria using transduction. If the Cosmids contain, for example, genes for resistance against antibiotics, the transfected bacteria are then able to survive and to spawn in a nutrient solution containing the antibiotic and can thus be selected. Cosmids can be used to build genomic libraries.