* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download DNA Replication
DNA barcoding wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
Gene expression wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Holliday junction wikipedia , lookup
DNA sequencing wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Molecular evolution wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Biosynthesis wikipedia , lookup
Community fingerprinting wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Molecular cloning wikipedia , lookup
Non-coding DNA wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
DNA REPLICATION Understandings: • Nucleosomes help to supercoil the DNA. • DNA structure suggested a mechanism for DNA replication. • DNA polymerases can only add nucleotides to the 3’ end of a primer. • DNA replications is continuous on the leading strand and discontinuous on the lagging strand. • DNA replication is carried out by a complex system of enzymes. • Some regions of DNA do not code for proteins but have other important functions. Applications and Skills • Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing. • Tandem repeats are used in DNA profiling. • Analysis of results of the Hershey and Chase experiment providing evidence that DNA is the genetic material Semi-Conservative Replication of DNA ■ The replication of DNA is semi-conservative and depends on complementary base pairing. ■ The two strands of DNA separate (by breaking the hydrogen bonds). ■ These two strands will serve as a template to create a complementary strand. ■ The newly synthesized strand will contain an original strand and new copy. Alternative Theories: ■ Conservative replication – both strands of the parent DNA remain together and another molecule is produced. ■ Dispersive replication – Every molecule produced by DNA replication has a mixture of old and new sections in both of its strands. Meselson and Stahl’s Experiments ■ They cultured E.coli bacteria for many generations in a medium where only nitrogen source (15N), so the nitrogen bases of the bacterial DNA was 15N. ■ Transferred to a less dense 14N medium. ■ Spun for 24 hours in a centrifuge. ■ DNA showed up as a dark band in UV light. Enzymes involved in DNA replication Gyrase ■ Relieves strain created by the unwinding of DNA by helicase. Helicase ■ Unwinds and separates the double-stranded DNA (creates a replication fork). Single Stranded Binding Proteins ■ Prevents separated strand from re-annealing. DNA Primase ■ Generates a short RNA sequence (primer) to initiate DNA synthesis. DNA Polymerase III ■ Extends new strands in a 5’ 3’ direction by joining nucleotides together. Okazaki Fragments ■ The lagging strand is copied away from the replication fork in short fragments. DNA Polymerase I ■ Removes RNA primers on lagging strand and replaces with DNA nucleotides DNA Ligase ■ Joins Okazai fragments together with phosphodiester bonds. ■ Stage 1: DNA gyrase moves in before helicase and relives strains in the DNA molecule. ■ Stage 2: Helicase uncoils the DNA double helix and splits it into two template strands. ■ Stage 3: DNA polymerase III adds nucleotides in a 5’ to 3’ direction. (On the leading strand it moves in the same direction as the replication fork) ■ Stage 4: DNA primase adds a short length of RNA attached by base pairing to the template strand of DNA. ■ Stage 5: DNA polymerase III starts replication next to the RNA primer and adds nucleotides in a 5’ to 3’ direction. (on the lagging strand, it moves away from the replication fork). ■ Stage 6: Short lengths of DNA are formed between RNA primers on the lagging strand, called Okazaki fragments. ■ Stage 7: DNA polymerase I removes the RNA primer and replaces it with DNA. A nick is left in the sugar-phosphate backbone of the molecule where two nucleotides are still unconnected. ■ Stage 8: DNA ligase seals up the nick by making another sugar-phosphate bond. Stages in DNA replication The leading and lagging strand ■ Because the two strands of the DNA double helix are arranged in an anti-parallel fashion, synthesis on the two strands occurs in very different ways. ■ Leading strand: is made continuously following the fork as it opens. ■ Lagging strand: is made in fragments moving away from the replication fork. – New fragments are created on the lagging strand as the replication fork exposes more of the template strand. – These fragments are called Okazaki fragments. Direction of Replication ■ DNA replication begins at sites called origins of replication. ■ In prokaryotes there is one site, in Eukaryotes there is multiple. ■ Replication occurs in both directions away from the origin. ■ The results appear as a replication bubble. ■ Free nucleotides (deoxynucleoside triphosphate) are added to the 3’ end. Non-coding regions ■ Majority of the human genome is comprised of non-coding DNA ■ Only 1.5% of our genes code for proteins. ■ Examples: – Telomeres – Regions of repetitive DNA at the end of a chromosomes, protects against chromosomal deterioration – Introns – Non-coding sequences within genes, are removed by RNA splicing prior to the formation of mRNA. – ncRNA genes – codes for RNA molecules that are not translated into proteins, example: genes for tRNA – Gene regulatory sequences (gene expression) – sequences that are involved in the process of transcription, includes promoters, enhancers and silencers What if we want to stop replication…. DNA Sequencing ■ Nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation for base sequencing. ■ DNA sequencing refers to the process by which the base order of a nucleotide sequence is elucidated. ■ The most widely used for DNA sequencing involves the use of chain-terminating dideoxynucleotides. Dideoxynucleotides ■ Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group necessary for forming a phosphodiester bond. ■ ddNTPs prevent further elongation of a nucleotide chain and effectively terminate replication. ■ The resulting length of a DNA sequence will reflect the specific nucleotide position at which the ddNTP was incorporated. Sequencing Tandem repeats ■ A variable number of tandem repeat is a short nucleotide sequence that shows variation between individuals in terms of the number of times the sequence is repeated. ■ Can be inherited as an allele. ■ Used in DNA profiling. Nucleosomes ■ Nucleosomes help to supercoil DNA. ■ Eukaryotic DNA is associated with proteins called histones. ■ Histones help package the DNA into structures called nucleosome. ■ Nucleosome consist of a central core of eight histone protein with DNA coiled around the proteins. ■ Supercoiling allows a great length of DNA to be packed into a much smaller space within the nucleus.