DNA Structure

DNA Backbone alternating sugar-phosphate backbone sugar and phosphate backbone attached by a phosphodiester bond DNA is said to be read from 5’ to 3’ opposing backbones are antiparallel 5’ 3’ 1’ 2’ 4’ Antiparallel = upside down 5’  3’ labeled similarly to proteins with numbered carbons (α1-4), except we use prime for DNA.

Nitrogenous Bases

Complementary Base Pairing Chargaff’s Rule adenine : thymine (1:1) guanine : cytosine (1:1) the diameter of a base pair is 2 nm 3 H-bonds between G&C 2 H-bonds between A&T 2 nm = 2 nanometers

Overall DNA Structure right handed helix 0.34 nm between adjacent base pairs one turn of the helix is 10 base pairs (3.4 nm) A nm = one thousand-millionth of a m. It can be written as 1 m / 1,000,000,000. 0.34 nm 3.4 nm

Overall DNA Structure minor groove major groove Major and Mionor groove because DNA molecule is not a perfect helix. Consider a spiral stair case What makes it uniform in diameter? The support beam down the centre. There is no “support beam” down the centre of a DNA molecule.

Three DNA Types A-DNA B-DNA Z-DNA

Three DNA Types 1 angstrom = 1.0 × 10-10 meters A-DNA B-DNA Z-DNA helical rotation right-handed left-handed # base pairs per 360° 10.7 10 12 helical diameter 25.5 Å 23.7 Å 18.4 Å major/minor grooves yes no – all grooves very similar in height Don’t need to memorize. A and B DNA are found naturally in our bodies. Z DNA reverse direction, or left handed. Much less common.

DNA Structure - Review

DNA Replication

DNA Replication All cells are capable of giving rise to a new generation of cells through DNA replication (copying) and cell division. Mitosis: replicated DNA in nucleus is divided equally between two daughter cells

1958 – Meselson & Stahl http://www.youtube.com/watch?v=JcUQ_TZCG0w grew E. coli in 15N media 14N is the usual isotope; 15N is heavier ensured that the bacterial DNA only contained 15N

Dark Blue = parental strand (original DNA) Figure 16.10 a–c The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple- mentary strand. Each strand of both daughter mol- ecules contains a mixture of old and newly synthesized DNA. Parent cell First replication Second CONSERVATIVE MODEL Dark Blue = parental strand (original DNA) Light Blue = new strand SEMI-CONSERVATIVE MODEL DISPERSIVE MODEL

Meselson & Stahl Experiment DNA was isolated and its weight determined through centrifugation

DNA Replication DNA replicates in a semi-conservative manner one strand of DNA is used as a template for the other strand 5’ A T G T C A G 3’ 3’ T A C A G T C 5’

3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (b) The first step in replication is separation of the two DNA strands. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. A C T G Figure 16.9 a–d 5’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’

DNA Replication DNA replication starts at a number of sites along a chromosome – origins of replication dozens of proteins are involved in the process of DNA replication

DNA Replication Initiation DNA helicase – unwinds DNA to break its bonds single-stranded binding proteins (SSBPs) – bind to each of the single stranded pieces of DNA and keeps them seperated if there were nothing to keep the strands apart, they would reanneal (stick back together) DNA Replication Animation http://www.youtube.com/watch?v=I9ArIJWYZHI

Proteins / Enzymes of DNA Replication as DNA is being unwound by helicase, DNA in front of the helicase gets bunched up DNA gyrase – enzyme that loosens the tension in front of the replication fork DNA gyrase – shoe lace demo (prevents coil from unwinding too much or getting too tight) DNA polymerase III – main enzyme of DNA replication, called III simply because it was discovered after DNA polymerase I. RNA primer – needed to start relipcation 19

Replication Structures replication occurs in both directions from the origin of replication replication fork – structure generated by DNA helicase and SSBPs replication bubble – two replication forks which are close in proximity

DNA Replication Part 2

DNA Polymerase DNA polymerase – enzyme which synthesizes nucleotide chains in prokaryotes: DNA polymerase I, II, III, IV & V in eukaryotes: over 15 different types nucleotide chains only form in the 5’  3’ directionn (can only add to the 3’ end of the new strand) DNA polymerase III is primarily responsible for DNA replication in prokaryotes

DNA Polymerase III Substrates DNA template deoxyribonucleoside triphosphates (dNTPs) RNA primer 3’ 5’ 5’ 3’ 3’ 5’

ATP (Adenosine Triphosphate) ADENINE 3 PHOSPHATES RIBOSE

3’ Figure 16.13 New strand Template strand 5 end 3 end Sugar A T Base C G P OH Pyrophosphate 2 P Phosphate 3’ DNA Polymerase deoxyribonucleoside triphosphates (dNTPs)

Step 1 Primase makes a short RNA primer on the exposed single-stranded DNA (ssDNA). 5’ 3’ 3’ 5’

Steps 2, 3, and 4 DNA polymerase III binds to the end of the RNA primer. The appropriate nucleoside triphosphate binds to the polymerase. A pyrophosphate group (PPi, a diphosphate group originally isolated by heating phosphates) is cleaved, while the nucleotide is added to the end of the nucleotide chain. TTP P P T A A

3’ Figure 16.13 New strand Template strand 5 end 3 end Sugar A T Base C G P OH Pyrophosphate 2 P Phosphate 3’ DNA Polymerase deoxyribonucleoside triphosphates (dNTPs)

Leading Strand DNA polymerase III 3’ SSBPs 5’ 3’ primase gyrase helicase 5’ 3’ The leading strand is synthesized continuously during DNA replication 5’

Lagging Strand 5’ 3’ 3’ 5’ 3’ Okazaki fragment 5’

Connecting Lagging Strands DNA polymerase I – removes the RNA primer occurs in the 5’  3’ direction DNA ligase – connects the sugar-phosphate backbone (by creating phosphodiester bonds) of Okazaki fragments Okazaki fragments are typically 1000 to 2000 nucleotides (NTs) in length

Replication Animation DNA replication occurs: continuously on the leading strand discontinuously on the lagging strand Replication Animation

Replication Overview - 1 helicase unwinds the double stranded DNA structure creating a replication fork the single stranded region of the replication fork are maintained by SSBPs gyrase relieves the tension ahead of the replication fork

Replication Overview - 2 two original parent strands serve as templates for the new daughter strands daughter strands are produced in one of two methods leading strand (continuous polymerization) lagging strand (discrete polymerization) 1000 – 2000 NTs Okazaki fragments joined together

Replication Overview - 3 primase begins each new daughter strand with a short RNA primer DNA polymerase III extends a DNA strand from the RNA primer DNA polymerase I removes the RNA primer AND fills it in with DNA DNA ligase joins the sugar-phosphate backbones of all adjacent DNA segments

Mistakes in DNA Replication less than 1 error in 107 (10 million) NTs exonuclease – enzymes which can cut out sections of nucleic acids DNA polymerase I and III have polymerase and exonuclease activity to fix mistakes

DNA Repair Figure 16.17 A thymine dimer distorts the DNA molecule. 1 A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. 2 Nuclease DNA polymerase 3 Repair synthesis by a DNA polymerase fills in the missing nucleotides. DNA ligase DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. 4 Figure 16.17

Homework helps prepare you for tests!!!! Homework will be checked each class: Pg. pg. 209 #1-4 pg. 216 #1-9 Pg. 223 #1-5, 7-8