Biochemistry Chapter 6 DNA and Biotechnology

nucleoside

five carbon sugar (pentose) bound to nitrogenous base via covalent linkage of the base to the C-1’ of the sugar

nucleotides

nucleoside with one or more phosphate groups bound to its 5’ carbon

purines

contain two rings in their structure

adenine, guanine

pyrimidines

contain one ring in their structure

cytosine, thymine, uracil

aromatic

follows four rules: planar, cyclic, conjugated, 4n+2 pi electrons

exceptional stability

Hückel’s rule

aromatic compounds must have 4n+2 (where n is any integer) pi electrons

Chargaff’s rules

total amount of A equals total amount of T, and total amount of G equals total amount of C; thus purines will be equal to pyrimidines overall

B-DNA

right handed helix form of DNA; most common form

Z-DNA

alternative DNA helix (as opposed to B-DNA), right-handed, turn every 4.6 nm and 12 bases within each turn; may be contributed to by high GC content or salinity; unstable and difficult to research

Denaturing

separation of two strands that compose the DNA helix resulting in two single stranded complementary sequences

Reannealing

joining of complementary single stranded DNA sequences through complementary hydrogen bonds between paring bases

probe DNA

DNA with a known sequence, used as a primer to create new daughter strands in PCR

Histone

group of positively charged (basic) DNA-associated proteins that compacts the DNA to form chromatin

Chromatin

histone-DNA complex

H2A, H2B, H3

histones that DNA coils around, forming a nucleosome

Nucleosome

Complex of DNA and histones H2A, H2B, and H3 that forms a “ball of yarn” complex

H1

Histone that seals DNA off as it enters and leaves the nucleosome, further compacting chromatin

Heterochromatin

compacted chromatin

Euchromatin

uncompacted chromatin

telomere

TTAGGG repeating end at 5' end of chromosomes, which cannot be replicated by DNA polymerase. The repeating structure allows for some degradation before genes are impacted

centromere

point of connection between sister chromatids that holds them together until microtubules pull them apart during anaphase--consists of highly constricted DNA with a high GC concentration

replisome / replication complex

specialized proteins that assist DNA polymerases including topoisomerases, helicases, and primases

origin of replication

point at which DNA unwinds to allow DNA polymerase to bind and thus replication

helicase

unwinds DNA, generating two single stranded template strands

ssDNA-binding proteins

proteins that bind ssDNA to prevent degradation during DNA synthesis

nuclease

enzyme that degrades DNA

topoisomerase

prevent supercoiling (and thus relieve torsional stress) by nicking the DNA strand ahead of the helicase

semiconservative replication

one of the strands of the new DNA double helix is the parent and the other is the daughter

Okazaki fragments

DNA fragments produced by DNA polymerase on the lagging strand

primase

synthesize short primer for DNA polymerase to begin replication

DNA Polymerase III

prokaryotic DNA polymerase

DNA polymerase alpha, delta, and epsilon

eukaryotic DNA polymerases that replicate the leading and lagging strands; delta also fills in the gaps when RNA primers are removed

DNA polymerase gamma

replicates mitochondrial DNA

DNA polymerase beta and epsilon

involved in DNA repair

sliding clamp

trimer composed of DNA polymerases delta and epsilon as well as PCNA protein to help strengthen interaction between DNA polymerase and template strand

Metastasis

ability of cancer cells to migrate to distant tissues via the bloodstream or lymphatic system

oncogenes

genes involved in cell cycle activation whose gain-of-function mutations lead to cancer phenotype; typically dominant with GOF

tumor suppressor genes

genes involved in cell cycle inhibition or DNA repair whose loss-of-funcion mutations contribute to a cancer phenotype; typically LOF mutated recessive

proofreading

ability of DNA polymerase to ensure correct hydrogen bonding is occurring between base pairs and thus determine whether or not the correct base has been added to the growing nucleic acid molecule

mismatch repair

occurs during G2 phase of cell cycle, between S and M phases

regulated by genes MSH2 and MLH1 (MutS and MutL in prokaryotes)

Nucleotide excision repair (NER)

excision endonuclease nicks phosphodiester backbone on both sides of thymine dimer (formed by excitement by UV light) and removes the section. DNA polymerase then fills in the gap by synthesizing DNA 5’ to 3’, then ligase seals

Base excision repair (BER)

glycosylase enzyme removes mutated bases, such as cytosine—>uracil mutation (easily detected as uracil should not be found in DNA), then an AP (apurinic/apyrimidic) endonuclease removes the abasic nucleotides from the nucleic acid. DNA polymerase then fills in the gap and DNA ligase reanneals the nicks

recombinant DNA

allows a DNA fragment from any source to be multiplied by either gene cloning or PCR

vector

recombinant viral or bacterial plasmids that can be transferred to a host bacterium after insertion of the DNA of interest

restriction enzymes (restriction endonucleases)

enzymes that recognize specific palindromic dsDNA sequences, such as 5’ GAATTC 3’. Some produce offset cuts, creating sticky ends

complementary DNA (cDNA)

DNA synthesized using a post-slicing mRNA template, sequenced to form expression libraries

Southern Blot

used to determine presence and quantity of DNA in a sample

Southern blot probe

known ssDNA sequence labeled with radioisotopes or indicator proteins to detect the presence of a desired sequence

dideoxyribonucleotide

deoxyribonucleotide with 3’ OH replaced with a 3’ H, preventing lengthening of the nucleic acid strand

transgenic mice

mice altered in the germ line by introducing a cloned gene into fertilized ova or into embryonic stem cells

can be developed by microinjecting cloned gene into nucleus of newly fertilized ovum or using embryonic stem cells, the latter of which allows for selection of cells with the transgene successfully inserted

transgene

cloned gene that is introduced into a transgenic organism

knockout mice

mice in which a target gene has been intentionally knocked out